P62 sqstm1

Cell lysates were harvested after treatments and time points indicated by using RIPA buffer (Sigma, St. Louis, MO) with protease inhibitor cocktails (Roche, Indianapolis, IN) following treatments and time points as indicated. Samples were boiled for 10 min at 95 °C, and they were resolved by SDS-PAGE. Membranes were blocked with 5% dry nonfat milk in TBS-Tween for 1 h at room temperature and probed with primary antibodies at the manufacturer’s recommended concentrations.
The primary antibodies used were PI3 kinase class III/VPS34 (#3811S, RRID: AB_2062856); Total ULK1 (#4773S, RRID: AB_2288252); Phospho-p44/42 MAP kinase (#9101S, RRID: AB_331646); p44/42 MAP kinase (Erk1/2) (#9102, RRID: AB: 330744); LC3 (#NB100–2220, RRID: AB_10003146) (Novus Biologicals, Littleton, CO); p62/SQSTM1 (#H00008878-M01, RRID: AB_437085) (Abnova). Anti-β-actin (#12262, RRID: AB_2566811) (Cell Signaling, Danvers, MA) was used as the protein-loading control. Secondary antibodies were purchased from Cell Signaling Technology including anti-rabbit IgG (#7074S, RRID: AB_2099233) and anti-mouse IgG (#7076S, RRID: AB_330924). The results of western blots were assessed by comparing the intensity of bands by using Image J (RRID: SCR_003070).

This study used reagents obtained from the commercial companies indicated below. Fetal bovine serum (FBS, Cat No. 12483-020) and TRIzol (GIBCO BRL, Gaithersburg, MD, USA); RPMI-1640 medium, Tween-20, bovine serum albumin (BSA), skim milk, and rapamycin (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA); Abs against molecules such as LAMP-2 (sc-18822), lamin B (sc-374015), PUMA (sc-377015), and actin (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Abs against molecules such as ATG5 (Cat No. 9164), ATG12 (Cat No. 4180), FoxO3a (Cat No. 12829), AMPKα (Cat No. 5832), phospho-AMPKα (Cat No. 2535), and Bax (Cat No. 2772), and horseradish peroxidase-conjugated anti-rabbit IgG (Cat No. 7074) and anti-mouse IgG (Cat No. 7076) (Cell Signaling Technology, Inc., Beverly, MA, USA); anti-LC3 antibody (Cat No. NB100-2220, Novus Biologicals, Saint Charles, MO, USA); p62/SQSTM1 (Cat No. H00008878-M01, Abnova, Taipei City, Taiwan); DyLight 549- and Alexa Fluor 488-conjugated secondary Abs (Thermo Fisher Scientific, Waltham, MA, USA); Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA, USA); compound C (HY-13418A, Medchemexpres, Monmouth, NJ, USA); bafilomycin A1 (Tocris Bioscience, Bristol, UK); 3-MA (Merckmillipore, Darmstadt, Germany)

Cells were trypsinized, washed well in PBS and pelleted before adding cold lysis buffer (Biosource International; Camarillo, CA) containing 1× protease and phosphatase inhibitors (Sigma-Aldrich). Quantification was done using BCA protein quantification kit (Thermo Fisher, Italy) as described by the manufacturer. Incubation for 30 min with reaction reagent was done at 37 °C to stimulate colorimetric reaction, and absorbance was then measured on VICTOR plate reader (486 nm). A total of 20 µg of proteins were loaded on 4–20% gradient gel and SDS-PAGE (Bio-Rad, Italy) was done as described in details elsewhere [17 (link)]. Primary antibodies used in the study were anti-: MCL1, PARP, total and phospho ERK, phospho AKT, phospho PI3K, phospho mTOR, BCL2, BCL-XL Caspase-3 (Cell Signaling), LC3, GAPDH, BECLIN 1, ATG5 (Novus Biologicals, Littleton, CO), p62/SQSTM1 (Abnova, Taipei City, Taiwan), PCNA (SCBT, Dallas, TX) using dilutions suggested by the manufacturer. Where necessary, a densitometry was done (ImageJ software from the National Institutes of Health; Bethesda, MD was used), using the expression of GAPDH for data normalization.