Anti gapdh ab9485

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The following primary and secondary antibodies were used to detect protein expression: anti-CPS1 (B1) (sc376190, 1:500) from Santa-Cruz Biotechnology (Santa Cruz, CA, USA), anti-actin (clone AC-40, dilution 1:1000), anti-CPS1 [EPR7493-3] (ab129076, 1:1000), anti-GAPDH (ab9485, 1:1000) from Abcam (Cambridge, UK), anti-SDHA (D6J9M) XP® (#11998, 1:1000), anti-Smac/Diablo (79-1-83) (#2954, 1:1000), anti-COX IV (#11967S, 1:200), anti-Hop (D10E2) (#5670, 1:1000), anti-Calnexin (C5C9) (#2679, 1:1000) from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies HRP-linked goat anti-rabbit (sc-2004, 1:5000) or goat anti-mouse (sc-2005, 1:5000) were from Santa-Cruz Biotechnology (Santa Cruz, CA, USA). Secondary goat anti-rabbit IgG H&L Alexa Fluor®488 (ab150077) was from Abcam (Cambridge, UK), and goat anti-mouse IgG H&L F(ab)₂ Alexa Fluor®594 (#8890S) was from Cell Signaling Technology (Danvers, MA, USA).

Kumatakenin (PubChem CID: 5318869) of ≥95% purity and sulfasalazine (SASP, PubChem CID: 5359476) were obtained from Sigma-Aldrich (St. Louis, MO, United States of America). Anti-enolase (Eno3, ab232759), anti-iron regulatory protein (IRP1, ab183721), anti-4-HNE (ab48506), and anti-GAPDH (ab9485) antibodies were obtained from Abcam (Hong Kong, China). Other reagents were purchased from Sigma-Aldrich.

Protein concentration was determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Having been separated by 10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), proteins were transferred onto the polyvinylidene difluoride membranes (PVDF, Millipore, Burlington, Massachusetts, USA) for 120 minutes. Then, cells were blocked with TBST including 5% nonfat skimmed milk and then incubated with primary antibodies (anti‐EZH2, ab186006, 1:1000; anti‐VEGFA, ab46154, 1 μg/mL; anti‐MMP2, ab97779, 1:2000; anti‐MMP9, ab119906, 1:1000; anti‐GAPDH, ab9485, 1:2500; Abcam, Cambridge, MA, USA). Subsequently, secondary antibodies were supplemented to culture the cells (Goat anti‐rabbit IgG H&L, ab6721, 1:10 000; Goat anti‐mouse IgG H&L, ab6789, 1:2000; Abcam). The signal detection was conducted by means of ECL system (Life Technology). GAPDH was regarded as an internal reference.

Cells were washed with PBS and then lysed with lysis buffer [1 M Tris, 2.5 M NaCl, 10% glycerol, 0.5 M glycerophosphate, 1% Tween 20, 0.5% NP-40 and a complete protease inhibitor tablet (Roche)] for 20 min on ice. The cell lysates were separated by 10 or 12% denaturing SDS-PAGE. The proteins were then transferred to nitrocellulose membranes (Whatman), blocked with PBS containing 5% milk and 0.05% Tween and incubated with specific primary antibodies overnight. After being washed with PBS containing 0.05% Tween, the blots were incubated with peroxidase-conjugated secondary antibodies labelled with horseradish peroxidase (HRP) (Sigma, Italy) and developed using ECL according to the manufacturer’s instructions. Rabbit polyclonal anti-GAPDH (ab 9485) and anti-procaspase-9 (ab 32068) were purchased from Abcam (Italy) and diluted 1:500. Rabbit anti-PARP (9542) and anti-p53 (Ser15-9286) were purchased from Cell Signaling Technology (Italy) and diluted 1:500. Anti-cleaved caspase-3 (9661), also purchased from Cell Signaling Technology, was diluted 1:1000. Rabbit anti-Bcl2 (ab 59348) and rabbit polyclonal anti-cyclin B1 (4138), purchased from Santa Cruz Biotechnology (Italy), were diluted 1:500. Rabbit anti-procaspase-8 (ab 49853) was purchased from Sigma and diluted 1:1000.

Total tissues or cellular RNA were obtained using TRIzol reagent (TaKaRa, Dalian, China), cDNA was synthesized using PrimeScript RT reagent Kit (TaKaRa, Dalian, China), and qRT-PCR was performed using SYBR Green Master Mix Reagen kit (GenStar, Beijing, China). U6 or GAPDH were synthesized to normalize the mRNA or protein level. All primers used for quantitative real-time PCR (qRT-PCR) are listed in Table 3.
The Western blot (WB) experiment was examined as supplementary data. The primary antibodies anti-CFL2 (ab14134) and anti-GAPDH (ab9485) were purchased from Abcam (Cambridge, UK), secondary antibody anti-immune rabbit IgG-HRP (LK2001) was purchased from Sungene Bio (Tianjin, China) and ECL luminous fluid (Solarbio, Beijing, China) was used to detect the antibody reacting bands.