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CBA039 Insulin Receptor (β-subunit) ELISA Kit

CBA039
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Detects and quantifies the levels of insulin receptor (IR) β-subunit independent of its phosphorylation status. The IR is a cell surface receptor that belongs to the superfamily of the growth factor receptor tyrosine kinases and regulates multiple signaling pathways. Although this kit is designed for use with human cell lines, it cross-reacts with mouse and rat cells.
      Catalogue NumberCBA039
      Brand Family Calbiochem®
      Materials Required but Not Delivered Plate reader capable of measurement at or near 450 nm.
      Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.)
      Cell extraction buffer (see Recommended Formulation, p. 4).
      Deionized or distilled H2O.
      Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      Graph paper: linear (Cartesian), log-log, or semi-log, as desired.
      Glass or plastic tubes for diluting and aliquoting standard.
      Absorbent paper towels.
      Calibrated beakers and graduated cylinders in various sizes.
      References
      ReferencesBevan, P. 2001. J. Cell. Sci. 114, 1429.
      Skorey, K.I., et al. 2001. Anal. Biochem. 291, 269.
      Ottensmeyer, F.P. et al. 2000. Biochemistry 39, 12103.
      Playford, M.P., et al. 2000. Proc. Nat'l. Acad. Sci. USA 97, 12103.
      Virkamaki, A., et al. 1999. J. Clin. Invest. 103, 931.
      Wei, L., et al. 1995. J. Biol. Chem. 270, 8122.
      Ebina, Y., et al. 1985. Cell 40, 747.
      Ullrich, A., et al. 1985. Nature 313, 756.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsIR (β-subunit) Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Positive controlIR (β-subunit)
      Applications
      Biological Information
      Assay range0.94-60 ng/ml
      Assay time4 h
      Sample TypeCells
      Physicochemical Information
      Sensitivity< 0.5 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Insulin Receptor (β-subunit) ELISA Kit is designed to detect and quantify levels of the insulin receptor β-subunit independent of its phosphorylation status. Both natural (heterotetrameric) and recombinant IR react in this assay. This ELISA is not cross-reactive with IGF-1R. Although this ELISA kit is developed using cells containing human insulin receptor, mouse and rat insulin receptor will cross react. This assay is intended for detection of IR from lysates of cells.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contentsof the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsIR (β-subunit) Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Specifications

      Documentation

      Insulin Receptor (β-subunit) ELISA Kit MSDS

      Title

      Safety Data Sheet (SDS) 

      Insulin Receptor (β-subunit) ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA039

      References

      Reference overview
      Bevan, P. 2001. J. Cell. Sci. 114, 1429.
      Skorey, K.I., et al. 2001. Anal. Biochem. 291, 269.
      Ottensmeyer, F.P. et al. 2000. Biochemistry 39, 12103.
      Playford, M.P., et al. 2000. Proc. Nat'l. Acad. Sci. USA 97, 12103.
      Virkamaki, A., et al. 1999. J. Clin. Invest. 103, 931.
      Wei, L., et al. 1995. J. Biol. Chem. 270, 8122.
      Ebina, Y., et al. 1985. Cell 40, 747.
      Ullrich, A., et al. 1985. Nature 313, 756.

      Brochure

      Title
      Protein Kinase Assay and Detection Kits Brochure
      User Protocol

      Revision19-October-2010 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      StorageUpon arrival store the entire contentsof the kit at 4°C.
      Intended useThe Calbiochem® Insulin Receptor (β-subunit) ELISA Kit is designed to detect and quantify levels of the insulin receptor β-subunit independent of its phosphorylation status. Both natural (heterotetrameric) and recombinant IR react in this assay. This ELISA is not cross-reactive with IGF-1R. Although this ELISA kit is developed using cells containing human insulin receptor, mouse and rat insulin receptor will cross react. This assay is intended for detection of IR from lysates of cells.
      BackgroundInsulin receptor (IR), a cell surface receptor, binds insulin and mediates its action on target cells. Insulin receptor belongs to the superfamily of the growth factor receptor tyrosine kinases that regulate multiple signaling pathways through activation of a series of phosphorylation cascades. The insulin receptor is a heterotetrameric membrane glycoprotein consisting of disulfide-linked subunits in a β-α-α-β configuration. The α-subunit (135 kDa) is completely extracellular, whereas the β-subunit (95 kDa) possesses a single transmembrane domain with tyrosine kinase activity. Insulin binding to the extracellular domain leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated. Once activated, the IR initiates a variety of metabolic functions including glucose transport, glycogen synthesis, protein synthesis, translational control and mitogenesis. Defects in the IR signaling pathway result in insulin resistance and thus high blood glucose associated with type II/non-insulin-dependent diabetes. The IR also plays an important role in neurological function and in hypertension. Important down-stream proteins associated with IR signaling include IRS-1, JAK1, JAK2, STAT1, STAT3, PI3 kinase, GSK-3β, PKC, SHC, ERK1/2, and many others. Insulin receptor and insulin-like growth factor 1 receptor (IGF-1R), which are structurally related, share conserved tyrosine residues that are phosphorylated in IR in response to insulin and in IGF-1R in response to IGF-1. Functionally, the insulin receptor regulates metabolism and IGF-1R mediates growth and differentiation. The catalytic loops within the tyrosine kinase domains of the IR/IGF-1R share the same residue sequence with a three-tyrosine motif corresponding to Tyr1158/1162/1163 (for the IR) and Tyr1131/1135/1136 (for the IGF-1R). It is generally believed that autophosphorylation within the activation loop proceeds in a progressive manner initiating at the second tyrosine (1162 or 1135), followed by phosphorylation at the first tyrosine (1158 or 1131), then the last (1163 or 1136), upon which the IR or IGF-1R becomes fully active.
      Principles of the assayThe Calbiochem® Insulin Receptor (β-subunit) Kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for IR (β-subunit) (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing IR, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the IR (β-subunit) antigen binds to the immobilized (capture) antibody. After washing, an antibody specific for IR (β-subunit) is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized IR (β-subunit) protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase-labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of IR (β-subunit) present in the original specimen.
      Materials provided• Human IR (β-subunit) (Kit Component No. JA8105-1EA): 2 vials, Refer to vial label for quantity and reconstitution volume
      • Standard Diluent Buffer (Kit Component No. JA8106-25ML): 1 bottle, Contains 15 mM sodium azide; 25 ml per bottle
      • IR (β-subunit) Antibody-Coated 96-Well Plate (Kit Component No. JA8107-1EA): 1 plate, 96 wells per plate
      • Rabbit Anti IR (β-subunit) Detection Antibody (Kit Component No. JA8108-11ML): 1 bottle, Contains 15 mM sodium azide; 11 ml per bottle
      • Anti-Rabbit IgG-Horseradish Peroxidase (HRP) Concentrate (Kit Component No. JA8109-125UL): 1 vial, (100x). Contains 3.3 mM thymol; 0.125 ml per vial
      • HRP Diluent (Kit Component No. JA8110-25ML): 1 bottle, Contains 3.3 mM thymol; 25 ml per bottle
      • Wash Buffer Concentrate (Kit Component No. JA8111-100ML): 1 bottle, (25x); 100 ml per bottle
      • Soluble Substrate (Kit Component No. JA8112-25ML): 1 bottle, Tetramethylbenzidine (TMB); 25 ml per bottle
      • Stop Solution (Kit Component No. JA8113-25ML): 1 bottle, 25 ml per bottle
      • Plate Sealers (Kit Component No. JA8114-1EA): 3 adhesive strips
      Materials Required but not provided Plate reader capable of measurement at or near 450 nm.
      Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.)
      Cell extraction buffer (see Recommended Formulation, p. 4).
      Deionized or distilled H2O.
      Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      Graph paper: linear (Cartesian), log-log, or semi-log, as desired.
      Glass or plastic tubes for diluting and aliquoting standard.
      Absorbent paper towels.
      Calibrated beakers and graduated cylinders in various sizes.
      Precautions and recommendations• Disposal Note: This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.
      When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.
      Plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediately reseal the bag and store at 4°C to maintain plate integrity.
      Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely and mix well prior to analysis.
      If particulate matter is present, centrifuge or filter prior to analysis.
      All standards, controls and samples should be run in duplicate.
      Samples containing IR (β-subunit) protein extracted from cells should be diluted with Standard Diluent Buffer at least 1:10. This dilution is necessary to reduce the matrix effect of the cell lysis buffer.
      When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells.
      Cover or cap all reagents when not in use.
      Do not mix or interchange different reagent lots from various kit lots.
      Do not use reagents after the kit expiration date.
      Read absorbances within 2 h of assay completion.
      In-house controls should be run with every assay. If control values fall outside pre-established ranges, the accuracy of the assay is suspect.
      All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.
      Because Soluble Substrate is light sensitive, avoid prolonged exposure to light. Also avoid contact between Soluble Substrate and metal, or color may develop.
      All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.
      Washing Guidlines: Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided. Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into the bottom of each well. Take care not to scratch the inside of the well. After aspiration, fill the wells with at least 0.4 ml of diluted wash solution. Let soak for 15 to 30 s, then aspirate the liquid. Repeat as directed under Detailed Protocol. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. If using an automated washer, the operating instructions for washing equipment should be carefully followed. If your automated washer allows, 30 s soak cycles should be programmed into the wash cycle.
      Preparation• Cell Lysis Buffer: • 10 mM Tris, pH 7.4 • 100 mM NaCl • 1 mM EDTA • 1 mM EGTA • 1 mM NaF • 20 mM Na4P2O7 • 2 mM Na3VO4 • 1% Triton® X-100 detergent • 10% glycerol • 0.1% SDS • 0.5% deoxycholate • 1 mM PMSF (stock is 0.3 M in DMSO) • Protease Inhibitor Cocktail Set III (Cat. No. 539134) This buffer is stable for 2-3 weeks at 4°C or for up to 6 months when aliquoted (without protease inhibitors and PMSF added) and stored at -20°C. When stored frozen, the Cell Extraction Buffer should be thawed on ice. Important: add the protease inhibitors just before using. The stability of protease inhibitor supplemented Cell Extraction Buffer is 24 h at 4°C. PMSF is very unstable and must be added prior to use, even if added previously. • Preparation of Cell Lysate: This protocol has been successfully applied to several cell lines. Researchers should optimize the cell lysate procedures for their own applications. 1. Collect cells in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent). 2. Wash cells twice with cold PBS. 3. Remove and discard the supernatant and collect the cell pellet. (At this point the cell pellet can be frozen at -80°C and lysed at a later date). 4. Lyse the cell pellet in Cell Lysis Buffer for 30 min, on ice, with vortexing at 10 min intervals. The volume of Cell Lysis Buffer depends on the cell number in cell pellet and expression of IR. 5. Transfer extract to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate to clean microfuge tubes. These samples are ready for assay. Lysates can be stored at -80°C. Avoid multiple freeze/thaw cycles.
      Reagent preparation• Reconstitution and Dilution of IR Standard: Note: This IR standard is a lyophilized lysate from human IR-transfected CHO cells (CHO-T). The standard is calibrated against the mass of purified recombinant IR (β-subunit) expressed in E. coli. 1. Reconstitute IR Standard with Standard Diluent Buffer. Refer to standard vial label for instructions. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 60 ng/ml IR. Use standard within 1 h of reconstitution. 2. Add 0.25 ml of Standard Diluent Buffer to each of 6 tubes labeled 30, 15, 7.5, 3.75, 1.87 and 0.94 ng/ml IR. 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps.

      Table 1: Dilution of IR (β-subunit) Standard

      Discard all remaining reconstituted and diluted standards after completing assay. Return the Standard Diluent Buffer to the refrigerator.

      • Storage and Final Dilution of Anti-Rabbit IgG-peroxidase (HRP): Please Note: The Anti-Rabbit IgG-HRP Concentrate is in 50% glycerol. This solution is viscous. To ensure accurate dilution, allow Anti-Rabbit IgG-HRP Concentrate to reach room temperature. Gently mix. Pipette Anti-Rabbit IgG-HRP Concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. 1. Dilute 10 µl of this 100X concentrated solution with 1 ml of HRP Diluent for each 8-well strip used in the assay. Label as Anti-rabbit IgG-HRP Working Solution.

      Table 2: Sample Dilutions

      2. Return the unused Anti-Rabbit IgG-HRP Concentrate to the refrigerator. • Dilution of Wash Buffer: Allow the 25X concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. Dilute 1 volume of the 25X wash buffer concentrate with 24 volumes of deionized water (e.g., 50 ml may be diluted up to 1.25 liters, 100 ml may be diluted up to 2.5 liters). Label as Working Wash Buffer. Store both the concentrate and the Working Wash Buffer in the refrigerator. The diluted buffer should be used within 14 days.
      Detailed protocolBe sure to read the Precautions and Recommendations section before carrying out the assay. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.

      Note: A standard curve must be run with each assay.

      1. Determine the number of 8-well strips needed for the assay. Insert these in the frame(s) for current use. (Re-bag extra strips and frame. Store these in the refrigerator for future use.)
      2. Add 100 µl of the Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl of standards to the appropriate wells. Samples prepared in Cell Extraction Buffer must be diluted 1:10 or greater in Standard Diluent Buffer (for example, 10 µl sample into 90 µl buffer). While a 1:10 sample dilution has been found to be satisfactory, higher dilutions such as 1:50 or 1:100 may be optimal. Tap gently on side of plate to thoroughly mix. (See Reagent Preparation, Section B.)
      4. Cover plate with plate sealer and incubate for 2 h at room temperature.
      5. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for washing.
      6. Pipette 100 µl of anti-IR (β-subunit) (Detection Antibody) solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      7. Cover plate with plate sealer and incubate for 1 h at room temperature.
      8. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      9. Add 100 µl Anti-Rabbit IgG-HRP Working Solution to each well except the chromogen blank(s). (Prepare the working dilution as described in Reagent Preparation, Section C.)
      10. Cover plate with the plate sealer and incubate for 30 min at room temperature.
      11. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for washing
      12. Add 100 µl of Soluble Substrate to each well. The liquid in the wells will begin to turn blue.
      13. Incubate for 30 min at room temperature and in the dark. Please Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for chromogen substrate is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (Abs) of 2.0. The Abs values should be monitored and the substrate reaction stopped before the Abs of the positive wells exceed the limits of the instrument. The Abs values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 2.0 Abs units, stopping the assay after 20 to 25 min is suggested.
      14. Add 100 µl of Stop Solution to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow.
      15. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µl each of Soluble Substrate and Stop Solution. Read the plate within 2 h after adding the Stop Solution.
      16. Plot on graph paper the absorbance of the standards against the standard concentration. (Optimally, the background absorbance may be subtracted from all data points, including standards, unknowns and controls, prior to plotting.) Draw the best smooth curve through these points to construct the standard curve. If using curve fitting software, the four parameter algorithm provides the best curve fit.
      17. Read the IR (β-subunit) concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply value(s) obtained for sample(s) by dilution factor to correct for the dilution in step 3. (Samples still producing signals higher than the highest standard (60 ng/ml) should be further diluted in Standard Diluent Buffer and re-analyzed, multiplying the concentration found by the appropriate dilution factor.)
      Standard curve

      Table 3: Typical Data Obtained with Standard

      The data was obtained for the various standards over the range of 0 to 60 ng/ml IR.

      Limitations of the assayDo not extrapolate the standard curve beyond the 60 ng/ml standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >60 ng/ml with Standard Diluent Buffer; re-analyze these and multiply results by the appropriate dilution factor.

      The influence of various lysate buffers has not been thoroughly investigated. The rate of degradation of native IR (β-subunit) in various matrices has not been investigated. Although IR (β-subunit) degradation in the Cell Lysis Buffer described in this protocol has not been seen to date, the possibility of this occurrence cannot be excluded.
      Sensitivity< 0.5 ng/ml
      Sensitivity NotesThe analytical sensitivity of this assay is 0.5 ng/ml of IR (β-subunit). This was determined by adding two standard deviations to the mean Abs obtained when the zero standard was assayed 30 times. The sensitivity of this ELISA was compared to immunoblotting using cell lysates with known quantities of IR (β-subunit). The data, presented in Figure 1, show that the sensitivity of the ELISA is approximately 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti-IR (β-subunit) antibody and chemiluminescent detection.

      Figure 1: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using cell lysates with known quantities of IR (β-subunit). The data, presented in Figure 1, show that the sensitivity of the ELISA is approximately 2X greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti-IR (β-subunit) antibody and chemiluminescent detection.

      Assay Range0.94-60 ng/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known IR (β-subunit) concentration were assayed in replicates of 16 to determine precision within an assay.


      Table 5: Inter-Assay Precision

      Samples were assayed 48 times in multiple assays to determine precision between assays.

      RecoveryThe recovery of IR (β-subunit) added to Jurkat cells lysate, adjusted to 200 µg/ml, averaged 102% when diluted in Standard Diluent Buffer.
      Parallelism

      Figure 2: IR (β -subunit) parallelism

      Natural IR was prepared from a lysate of human IR-transfected CHO cells and serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the IR standard curve. Parallelism was demonstrated by the figure below and indicated that the Standard accurately reflects IR (β-subunit) content in samples.

      LinearityHuman IR transfected CHO cells were grown in tissue culture medium containing 10% fetal calf serum and lysed with Cell Lysis Buffer. This lysate was adjusted to 60 ng/ml IR (β-subunit) and serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99 in both cases.

      Table 6: Linearity of Dilution

      Human IR transfected CHO cells were grown in tissue culture medium containing 10% fetal calf serum and lysed with Cell Lysis Buffer. This lysate was adjusted to 60 ng/ml IR (β-subunit) and serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99 in both cases.

      SpecificityThe Insulin Receptor (β-subunit) ELISA Kit is specific for measurement of IR (β-subunit) protein, regardless of phosphorylation state of insulin receptor.

      Figure 3: Specificity of IR (β-subunit) ELISA for IR

      In the experiments presented in Figure 2, IR CHO-T cells were stimulated using 100 nM insulin for 10 min. Unstimulated cells were used as control. Cell lysates from the cells were measured for the levels of IR and phosphorylated IR. The results show that Insulin Receptor (β-subunit) ELISA Kit detects phosphorylated IR in insulin-stimulated CHO-T and non-phosphorylated IR in unstimulated control cells.


      Figure 4: IR and IR phospho ELISAs

      Figure 4 shows that IR phosphorylation in CHO-T cells is dependent on the levels of insulin stimulation. CHO-T cells were treated with insulin at varying concentrations (0-100 nM), lysed and quantitated in parallel for contents of IR (β-subunit) and phosphorylated IR. The amount of IR (β-subunit) remains relatively constant, while levels of IR phosphorylation at (Tyr1158/1162/1163) decrease with diminishing insulin dose.

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