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  • WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. 24954904

    Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12-5-16L1 complex. The molecular mechanisms of Atg12-5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12-5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12-5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.
    Document Type:
    Reference
    Product Catalog Number:
    MABC91
    Product Catalog Name:
    Anti-Atg18 (WIPI-2) Antibody, clone 2A2

  • Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. 26150155

    Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death. 22421968

    TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4400
    Product Catalog Name:
    Anti-Renilla Luciferase Antibody, clone 5B11.2

  • Enrichment of GABARAP relative to LC3 in the axonal initial segments of neurons. 23671684

    GABAA receptor-associated protein (GABARAP) was initially identified as a protein that interacts with GABAA receptor. Although LC3 (microtubule-associated protein 1 light chain 3), a GABARAP homolog, has been localized in the dendrites and cell bodies of neurons under normal conditions, the subcellular distribution of GABARAP in neurons remains unclear. Subcellular fractionation indicated that endogenous GABARAP was localized to the microsome-enriched and synaptic vesicle-enriched fractions of mouse brain as GABARAP-I, an unlipidated form. To investigate the distribution of GABARAP in neurons, we generated GFP-GABARAP transgenic mice. Immunohistochemistry in these transgenic mice showed that positive signals for GFP-GABARAP were widely distributed in neurons in various brain regions, including the hippocampus and cerebellum. Interestingly, intense diffuse and/or fibrillary expression of GFP-GABARAP was detected along the axonal initial segments (AIS) of hippocampal pyramidal neurons and cerebellar Purkinje cells, in addition to the cell bodies and dendrites of these neurons. In contrast, only slight amounts of LC3 were detected along the AIS of these neurons, while diffuse and/or fibrillary staining for LC3 was mainly detected in their cell bodies and dendrites. These results indicated that, compared with LC3, GABARAP is enriched in the AIS, in addition to the cell bodies and dendrites, of these hippocampal pyramidal neurons and cerebellar Purkinje cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3420
    Product Catalog Name:
    Anti-Tau-1 Antibody, clone PC1C6