Mouse anti flag tag

Co-IP was performed to confirm the interaction between two proteins. The cellular protein was extracted from H9c2 cells using RIPA lysis buffer supplemented with 1% PMSF (Beyotime Biotech). After protein concentration determination, Co-IP was performed using a Co-IP kit (Thermo Scientific) according to the manufacturer’s instruction. Briefly, the antibody against GSK3β (ProteinTech) or flag tag (ProteinTech) was immobilized to AminoLink resin by incubating in sodium cyanoborohydride solution (75 mM), and the resin was pre-cross-linked to spin column. The protein samples were incubated with the antibody-resin complex in the tube for immunoprecipitation. Subsequently, the immunoprecipitate was eluted with elution buffer, and used for western blot to detect the content of GSK3β, WSB1, ubiquitin and GAPDH. The information of antibodies was shown as follows: rabbit anti-GSK3β (1:5000; ProteinTech), rabbit anti-WSB1 (1:1000; Thermo Scientific), rabbit anti-ubiquitin (1:5000; ProteinTech), mouse anti-flag tag (1:5000; ProteinTech), rabbit anti-HA tag (1:5000; ProteinTech), goat anti-rabbit IgG-HRP (1:3000; Solarbio), goat anti-mouse IgG-HRP (1:3000; Solarbio).

Cells were washed with phosphate-buffered saline (PBS) and lysed in 500 μl RIPA buffer with 5 μl protease inhibitor cocktail (100X, Merck, USA) on ice for 15 min, then centrifugation at 13 000 g for 10 min. The proteins in 20 μl supernatant were separated with 12% SDS-PAGE and transferred onto a nitrocellulose membrane. Then the membrane was incubated with rabbit anti-human primary antibody PAK4 (1:500–1:1000, Abcam, UK), mouse Anti-Flag Tag (1:1000–1:10 000, Proteintech, USA) and mouse anti-human primary antibody GAPDH (1:5000, KangChen Bio-tech Inc. China) at 4 °C overnight, respectively. Next, it was incubated with a horseradish peroxidase-labeled (HRP-labeled) goat anti-rabbit or anti-mouse secondary antibody (1:2000, Jackson Immunotech, UK) at room temperature for 1 h. The membrane was developed using enhanced chemiluminescence (ECL, Millipore, Germany) for testing.

The following antibodies were used in this study: mouse anti-ATF6 (Proteintech No. 66563-1-Ig), rabbit anti-α-subunit of GNPTAB (Abclonal Technology No. A15895), mouse anti-β-actin (Sigma No. A1978), mouse anti-clathrin heavy chain (BD Transduction Laboratories No. 610499), rabbit anti-CYP51A1 (Proteintech No. 13431-1-AP), rabbit anti-EGFP (Proteintech No. 50430-2-AP), mouse anti-FDFT1 (Santa Cruz Biotechnology No. sc-271602), mouse anti-Flag tag (Proteintech No. 66008-3-Ig), mouse anti-GAPDH (Proteintech No. 60004-1-Ig), mouse anti-GM130 (BD Transduction Laboratories No. 610823), mouse anti-lamin B1 (Proteintech No. 66095-1-Ig), mouse anti-lyso-bis-phosphatidic acid (LBPA) (Echelon Biosciences No. Z-PLBPA), rabbit anti-Myc tag (Proteintech No. 16286-1-AP), mouse anti-squalene epoxidase (Santa Cruz Biotechnology No. sc-271651). The polyclonal antibody against LDLR, the monoclonal antibody against HMGCR (A9), and the monoclonal antibody against SREBP2 (1D2) were prepared in our laboratory. The scFv (single-chain Fragment variable) M6P (mannose 6-phosphate) antibody was a kind gift from Dr. Thomas Braulke (University Medical Center Hamburg-Eppendorf, Germany). Primary antibodies were used at the dilution of 1:500 for immunofluorescent staining and 1:1,000 for immunoblotting.