Anti c myc 10828 1 ap

The total protein of HaCaT cells was extracted with a Total Protein Extraction Kit (Millipore, Burlington, MA, USA). The protein concentration was determined using a bicinchoninic acid (BCA) method with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% (w/v) skimmed milk at room temperature for 1 h, protein samples were incubated with the corresponding primary antibody at 4°C overnight. The following antibodies were used: anti-β-catenin (51067-2-AP; Proteintech, Wuhan, China), anti-c-Myc (10828-1-AP, Proteintech), and anti-VEGF (66828-1-Ig, Proteintech). Then, protein samples were incubated with the secondary antibody. Band signals were visualized by the enhanced chemiluminescence (ECL, Bio-RAD, Hercules, CA, USA) method according to the manufacturer's instructions. Relative protein expression was normalized to actin.

To evaluate protein expression the following primary antibodies were used: rabbit polyclonal anti-phospho-eIF2α (Ser51) (9721; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-eIF2α (9722; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-XBP1s (24868-1-AP; Proteintech, Rosemont, IL, USA), anti-BiP/GRIP78 (11587-1-AP; Proteintech), rabbit polyclonal anti-Caspase 9 (10380-1-AP; Proteintech, Rosemont, IL, USA), rabbit polyclonal anti-c-Myc (10828-1-AP; Proteintech, Rosemont, IL, USA), mouse monoclonal anti-γ-H2AX (Ser 139) (sc-517348; Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-BRCA-1 (OP92; EMD Millipore, Burlington, MA, USA), mouse monoclonal anti-RAD51 (G-9) (sc-377467; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit monoclonal anti-PARP (46D11) (9532; Cell Signaling, Danvers, MA, USA). Mouse monoclonal anti-β-actin (A5316; Sigma-Aldrich, Burlington, MA, USA) was used as loading control. The goat anti-Mouse IgGP Peroxidase Conjugate (401215; Sigma-Aldrich, Burlington, MA, USA) and the goat anti-Rabbit IgG Peroxidase Conjugate (DC03L; Sigma-Aldrich, Burlington, MA, USA) were used as secondary antibodies.

Western blotting assay were performed as described previously (Zang et al. 2022 (link)). The antibodies used were as follows: anti-MCCC2 (12117-1-AP, Proteintech, China); anti-ECHDC2 (bs-13049R, Bioss, China); anti-GLUT1 (21829-1-AP, Proteintech, China); anti-PKM2 (15822-1-AP, Proteintech, China); anti-SDHA (14865-1-AP, Proteintech, China); anti-G6PD (ab993, Abcam, UK); anti-c-Myc (10828-1-AP, Proteintech, China); anti-p-AKT (66444-1-lg, Proteintech, China); anti-P38 MAPK (14064-1-AP, Proteintech, China); anti-ubiquitin (10201-2-AP, Proteintech, China); anti-NEDD4 (21698-1-AP, Proteintech, China); anti-ITCH (20920-1-AP, Proteintech, China); anti-CBL (25818-1-AP, Proteintech, China); anti-GAPDH (10494-1-AP, Proteintech, China).

Cells were lysed on ice for 30 min in RIPA lysis buffer supplemented with protease inhibitors and a phosphatase inhibitor cocktail (Bimake), followed by centrifugation at 12,000 g for 10 min. The protein concentration was measured using a BCA Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The immunoblots were visualized by ECL chemiluminescence (GE healthcare, Buckinghamshire, UK) using a Bio-Rad gel image analysis system. The following primary antibodies were used in this study: anti-ETV4 (sc-113, Santa Cruz), anti-HK2 (22029-1-AP, Proteintech), anti-LDHA (19987-1-AP, Proteintech), anti-PDK1 (3062 T, Cell Signaling Technology), anti-c-MYC (10828-1-AP, Proteintech), anti-OCT4 (11263-1-AP, Proteintech), anti-NANOG (14295-1-AP, Proteintech), anti-LIN28 (11724-1-AP, Ptoteintech), anti-SHH (ab53281, Abcam), anti-GLI1 (66905-1-Ig, Proteintech), anti-CXCR4 (60042-1-Ig, Proteintech), anti-β-actin (A1978, Sigma-Aldrich).

Matrine (S2322, Selleck, USA) was dissolved in ddH₂O with a final concentration of 40 mg/ml and stored at +4°C. ddH₂O was used as vehicle control for Matrine. Other reagents and antibodies used included: Cycloheximide (CHX, S7418, Selleck, USA), MG132 (S2619, Selleck, USA), Actinomycin D (ACTD, S8964, Selleck, USA), cell counting kit-8 (CCK-8, CK04, Dojindo, Japan), Anti-c-Myc (10828-1-AP, Proteintech group, USA) and Anti-β-actin (23660-1-AP, Proteintech group, USA). DMSO as control for CHX, MG132, and ACTD.