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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: FEBS J. 2014 Jul 28;281(17):3816–3827. doi: 10.1111/febs.12905

Fig. 7. Effect of Sestrin2 and FIP200 deficiencies on p62 phosphorylation levels.

Fig. 7

(A,B) Physical association among endogenous Sestrin2, p62 and ULK1. HEK293 (A) and Sesn2+/+ and Sesn2−/− MEF (B) were treated with 100 μM etoposide for 16 hr to maximize endogenous Sestrin2 expression. Sestrin2 was immunopurified using protein G/A-conjugated anti-Sestrin2 antibody. Protein G/A-conjugated pre-immune IgG and empty protein G/A beads (−) were used as negative controls. Both anti-Sestrin2 immunocomplexes (IP) and whole cell lysates (WCL) were assayed through immunoblotting (IB). (C) Reduction of p62 Ser403 phosphorylation in Sestrin2-deficienct mouse embryonic fibroblasts (MEF). Whole cell lysates from wild-type (Sesn2+/+) and Sesn2−/− MEF were subjected to immunoblotting with indicated antibodies. (D) Induction of p62 Ser403 phosphorylation in FIP200-deficient MEF. Whole cell lysates from wild-type (Fip200+/+) and Fip200−/− MEF were subjected to immunoblotting with indicated antibodies. (E) Induction of basal p62 Ser403 phosphorylation in ULK1/2-deficient MEF was suppressed by BX-795, an inhibitor of TBK1. Wild-type (WT, Ulk1+/+/Ulk2+/+) and Ulk1−/−/Ulk2−/− (KO) MEF were treated with PBS (Con) or 10 μM BX-795 for 6 h, and subjected to immunoblotting with indicated antibodies. (F,G) Oligomycin-mediated energy depletion strongly induced Ser403 phosphorylation of p62 in WT MEF, but not as robustly in ULK1/2- or Sestrin2-deficient MEF (KO). WT (Ulk1+/+/Ulk2+/+ and Sesn2+/+ controls), Ulk1−/−/Ulk2−/− and Sesn2−/− MEF were treated with PBS (Con) or 10 μg/mL oligomycin for 6 h, and subjected to immunoblotting with indicated antibodies.