Fig. 5. TRIM32 is required for ULK1 activation through K63-linked polyubiquitin upon dexamethasone treatment.

(A) FLAG-TRIM32–expressing C2.7 cells differentiated for 3 days were treated with dexamethasone for 1 and 2 hours. Protein extracts were immunoprecipitated using an anti-FLAG antibody. Immunopurified complexes were analyzed by immunoblotting to detect the presence of TRIM32 and ULK1 using specific antibodies. (B) C2.7 cells differentiated for 3 days were treated with dexamethasone for 1 and 2 hours. Protein extracts were immunoprecipitated using an anti-ULK1 antibody. Immunopurified complexes were analyzed by immunoblotting to detect the presence of ULK1 and K63-linked polyubiquitin. An unrelated antibody was used as a negative control (IP CTR). (C) shCTR and shTrim32 C2.7 cells differentiated for 3 days were treated with dexamethasone for 2 hours. Protein extracts were immunoprecipitated using an anti-ULK1 antibody. Immunopurified complexes were analyzed by immunoblotting to detect the presence of ULK1 and K63-linked ubiquitin. An unrelated antibody was used as a negative control. Total extracts were probed with an anti-TRIM32 antibody to verify silencing efficiency. (D) BECLIN 1 overexpressing Trim32 WT and KO C2.7 cells were differentiated for 3 days and incubated with dexamethasone or nutrient-deprived medium (Starv) for 2 hours. Protein extracts were analyzed by immunoblotting to measure the levels of phospho–BECLIN 1 [pBCN 1 (Ser¹⁵)], BECLIN 1, and TRIM32. GAPDH was included as a loading control. (E) Trim32 WT and KO C2.7 cells differentiated for 3 days were incubated with dexamethasone for 2 hours. Cells were lysed, and protein extracts were analyzed by immunoblotting to measure the levels of phospho-ATG14 [pATG14 (Ser²⁹)], ATG14, and TRIM32. GAPDH was included as a loading control.