Fig. 4: P62 mediates selective autophagic degradation of TNC. | Nature Communications

Fig. 4: P62 mediates selective autophagic degradation of TNC.

From: Autophagy deficiency promotes triple-negative breast cancer resistance to T cell-mediated cytotoxicity by blocking tenascin-C degradation

Fig. 4

a Immunoblot of MDA-MB-231-WT or MDA-MB-231-Atg5KO#4 cells treated with EBSS for the indicated time points. b HEK293T cells were transfected with Flag-tagged p62, NDP52, NBR1, TAX1BP1, Tollip, OPTN or BNIP3L, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-TNC. c Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with FLAG-tagged wild-type p62 or its UBA ___domain deletion mutant, together with HA-tagged TNC. d Immunoprecipitation and immunoassay of extracts HEK293T cells transfected with Flag-p62, HA-TNC, and treated with EBSS for different hours. e Confocal microscopy of MDA-MB-231 cells treated with EBSS for 3 h or exposed to hypoxia for 12 h in the presence of BafA1. Scar bar, 10 µm. Line-scan analysis for each image is also shown. Green, TNC; Red, P62. f HEK293T cells were transiently transfected with p62 siRNA for 12 h, then co-transfected with HA-tagged TNC for another 48 h. Then the cells were treated with EBSS for different hours. g Construct deletion mutants of Flag-TNC according to the conserved domains of TNC. h HEK293T cells were transfected with Flag-tagged TNC or deletion mutants. Endogenous p62 was immunoprecipitated and the bound Flag-TNC proteins examined by immunoblot. All data are representative of three independent experiments.

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