Figure 2
CDK1 degradation involves p62/HDAC6-mediated selective autophagy. (A) MCF7 and HeLa cells were interfered with the indicated siRNAs, and extracts were blotted with the designated antibodies. (B) MCF7 and HeLa cells interfered with the indicated siRNAs were treated with MG132 4 hours before harvesting, and lysates were subjected to Western blot. (C) MCF7 and MCF10A cells were interfered and treated with etoposide (Eto) 24 hours before harvesting. Extracts were blotted with the indicated antibodies. (D) NP40 extracts from MCF7 cells treated or not with ammonium chloride (NH4Cl) for 24 hours were used to immunoprecipitate CDK1, and the obtained complexes were analyzed by immunoblotting. IP IgG: immunoprecipitation with normal mouse serum used as a control; Lys: Lysate from MCF7 cells untreated or treated with NH4Cl; K63-Ub: immunoblot using an anti-K63-ubiquitin polyclonal antibody that recognizes Lys63-linkage specific ubiquitination; Mw: molecular weight. Lanes are numbered from 1 to 6. (E) Co-immunoprecipitation experiments similar to (D) but using MCF7 cells treated with concanamycin A (Con A; 1 mg of extract protein) or 3-methyladenine (3-MA; 5 mg of extract protein) for 24 hours. (F) Extracts from exponentially growing MCF7 cells treated with NH4Cl, Con A or 3-MA for 24 hours were used to immunoprecipitate p62, or normal rabbit serum as a control (IgG). Immunocomplexes were analyzed by Western-blot. Lys: Lysates from MCF7 cells in the different conditions; Mw: molecular weight. Lanes are numbered from 1 to 6. Vertical white lines indicate that bands were taken from different exposures of the same anti-p62 blot. Full-length blots are given in Supplementary Fig. S2. (G) Comparison of p62, CDK1, and LC3 levels in MCF7 cells after the indicated treatments for 24 hours. C: Lysate from MCF7 untreated cells; NH4Cl: ammonium chloride; Con A: concanamycin A; 3-MA: 3-methyladenine. Quantitative fold change in CDK1 was determined relative to loading control.
