Anti p62 sqstm1

Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose transfer membranes. The blots were blocked with freshly prepared 5% nonfat milk in TBST for 1 h at room temperature. Then the blots were incubated at 4 °C overnight with primary antibodies. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated (HRP-conjugated) donkey anti-rabbit IgG or sheep anti-mouse IgG (Invitrogen, China) at room temperature for 1 h. ECL substrate (CLiNX, Shanghai, China) and ChemiScope Touch (CLiNX, Shanghai, China) were used for detecting HRP-conjugated antibodies. Primary antibodies including anti-P-AMPK (phospho S496), anti-P-S6K (phospho S424), and anti-P-AKT (Ser473) were brought from Abcam (China). Primary antibodies including anti-LC3, anti-P-ERK(Thr202/Tyr204), anti-PARP1, anti-vimentin, anti-SQSTM1/p62, anti-GAPDH, and anti-IGF2R(M6PR) were brought from Proteintech (China).

Antibodies used in this study were as follows: anti-Rab7 (Santa Cruz Biotechnology, sc-376362), anti-Rab7 (Proteintech, 55469-1-AP), anti-p53 (Santa Cruz Biotechnology, sc-126), anti-SQSTM1/p62 (Proteintech, 18420-1-AP), anti-LC3B (MBL, M186-3), anti-RILP (Proteintech, 13574-1-AP), anti-GDI2 (Proteintech, 10116-1-AP), anti-LAMP1 (Proteintech, 55273-1-AP), anti-MON1A (Proteintech, 23772-1-AP), anti-CD63 (Proteintech, 25682-1-AP), anti-CTSB (Proteintech, 12216-1-AP), anti-CTSD (Proteintech, 21327-1-AP), anti-EEA1 (CUSABIO, CSB-PA007405LA01HU), Anti-EEA1 mAb-Alexa Fluor^® 488 (MBL, M176-A48), anti-GAPDH (Proteintech, 60004-1-Ig), anti-MYC (Santa Cruz Biotechnology, sc-40), anti-FLAG (Biolegend, 637301), anti-GFP (Proteintech, 66002-1-Ig), anti-HA (Proteintech, 51064-2-AP), anti-GST (Proteintech, 10000-0-AP). Other reagents used in this study were: Hoechst 33342 (Sigma Aldrich, 14533), CQ (Sigma-Aldrich, C6628), Lyso-Tracker Red (Beyotime, C1046), and Lipofectamine® 2000 (Invitrogen, 11668019).

Human liver epithelial HepG2 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Gaithersburg, MD). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from HyClone (Logan, UT). Antibodies against cleaved cysteinyl aspartate specific proteinase (caspase)-9 (1:1000), cleaved caspase-3 (1:1000), Bcl2 (1:1000), Bax (1:1000), β-actin (1:1000), and microtubule-associated proteins 1A/1B light chain 3B (LC3B) (1:1000) were obtained from Cell Signaling Technology (Danvers, MA). Anti-poly ADP-ribose polymerase-1 (anti-PARP-1) (1:1000) was provided from Abcam (Cambridge, UK). Anti- SQSTM1/p62 (1:1000), anti-Beclin1 (1:1000), and anti-lysosomal-associated membrane protein 1 (anti-LAMP1) (1:1000) were purchased from Proteintech (Rosemont, IL). 3-methyladenine (3-MA) and chloroquine (CQ) were purchased from MedChem Express (Princeton, NJ). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and other chemical agents were of the analytical grade.

MAC-T cells were lysed in RIPA buffer containing a protease/phosphatase inhibitor cocktail on ice for 30 min.). Protein (equal amounts, 20 µg) were loaded on 10% or 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Roche). After blocking with 5% skim milk at 37°C for 1 h and the membranes were incubated with the following primary antibodies at 4°C overnight: anti-LC3 I/II (1:1000, #4108) and anti-cleaved-Caspase-3 (1:1000, #9664) from Cell Signaling Technology (Danvers, USA); anti-ATG5 (1:750, 10181-2-AP), anti-Beclin-1 (1:1000, 11036-1-AP), anti-p62/SQSTM1 (1:1000, 18420-1-AP), anti-ASC (1:1000, 10500-1-AP), anti-BAX (1:1000, 50599-2-Ig), anti-Bcl-2 (1:1000, 12789-1-AP), anti-Caspase-3 (1:1000), anti-PINK1 (1:1000, 23274-1-AP), anti-Parkin (1:1000, 14060-1-AP), anti-GAPDH (1:5000, 60004-1-AP) and anti-β-Actin (1:5000, 60008-1-AP) from Proteintech Group Inc (Rosemont, IL 60018, USA); anti-NLRP3 (1:500, AF2155) from Beyotime Biotechnology (Shanghai, China) and anti-Caspase-1 (1:1000, ab179515) from Abcam (Cambridge, UK). The immunoreactive bands were visualized with an ECL detection system (Tanon 6200 chemiluminescence imaging workstation, Tanon Science & Technology Co., Ltd. Shanghai, China). The protein bands were quantified by densitometry using ImageJ software (version 1.50).

Total protein separation and western blotting were performed as described previously [16 (link)]. Western blot analysis was performed using anti-cleaved PARP (#5625), anti-cleaved caspase-3 (#9661), anti-p-AMPK (Thr172) (#50081), anti-p-ERK1/2 (#4370), and anti-HA-Tag (#3724) antibodies purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3 (14600–1-AP), anti-P62/SQSTM1 (18420–1-AP), anti-phospho-Akt (Ser-473) (66444–1-Ig), anti-Akt (10176–2-AP), anti-mTOR (20657–1-AP), anti-P38 MAPK (14064–1-AP), anti-JNK (51151–1-AP), anti-ERK1/2 (16443–1-AP), anti-AMPK (10929–2-AP), anti-Nrf2 (16396–1-AP), anti-Keap1 (10503–2-AP), anti-FLAG-tag (66008–2-AP), anti-MYC-tag (60003–2-AP), and anti-β-actin (66009–1-Ig) antibodies were obtained from Proteintech Group Co., Ltd. (Wuhan, China). An anti-phospho-mTOR (Ser-2448) (ab109268) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-p-p38 (sc-7973), anti-p-JNK (sc-6254), and anti-Ub (sc-8017) antibodies were obtained from Santa Cruz Biotechnology., Inc. (Santa Cruz, CA, USA).

The primary antibodies were as follows: anti-LC3B (Abcam, ab-229327), anti-ATG5 (Bioss, bs-4005R), anti-Beclin 1 (Bioss, bs-1353R), and anti-GAPDH (Bioss, bs-0755R). Rabbit anti-DDDDK tag, anti-P62/SQSTM1, anti-MYC-tag, and mouse anti-GST tag antibodies were obtained from Proteintech. The secondary antibodies were goat anti-rabbit IgG/HRP (Bioss, bs-0259G-HRP) or goat anti-mouse IgG (H+L)/HRP (Bioss, bs-40296G-HRP).