Rabbit anti mmp9

The following antibodies were used: rabbit anti-MMP9 (Proteintech, cat.no.: 10375-2-AP); rabbit anti-Ki67 (Thermo Fisher Scientific Invitrogen, MA5-14520); rabbit anti-Gsta1 (Proteintech, cat.no.: 14475-1-AP); rabbit anti-Col3a1 (Proteintech, cat.no.: 22734-1-AP); rabbit anti-Tom20 (Proteintech, cat.no.: 11802-1-AP) and rabbit anti-SGLT2 (gift. Of H. Koepsell).

Cells were lysed on ice with Radio Immunoprecipitation Assay (RIPA) buffer containing 1 mM Phenylmethylsulfonyl fluoride (PMSF) to inhibit proteolysis. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF). The PVDF membranes were incubated with rabbit anti-MMP2 (1:1000; Proteintech, Cat. no.: 10373-2-AP), rabbit anti-Arg-1 (1:1000; CST, Cat. no.: 93668T), rabbit anti-MMP9 (1:1000; Proteintech, Cat. no.: 10375-2-AP), rabbit anti-VEGF (1:1000; Proteintech, Cat. no.: 19003-1-AP), rabbit anti-p65 (1:1000; Proteintech, Cat. no.: 10745-1-AP), rabbit anti-p-p65 (1:1000; CST, Cat. no.: 3033S), anti-AKT (1:1000; Proteintech, Cat. no.: 10176-2-AP), mouse anti-p-AKT (1:1000; Proteintech, Cat. no.: 66444-1-Ig), and mouse anti-β-actin (1:5000; Proteintech, Cat. no.: 60008-1-Ig) primary antibodies overnight at 4°C after blocking with 5% skim milk. Membranes were incubated with secondary antibodies for 2 h at room temperature, and proteins were visualized with an Ultra High Sensitivity ECL Kit.

The expression of TMEM40 protein in CC tissues and cell lines were evaluated with western blot. Briefly, the CC tissues and cells were lysed, and proteins were extracted through standard protocols. The proteins were separated by SDS–polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was blocked with 5% bovine serum albumin for 2 h. The immunoblots were incubated overnight at 4 °C with the following primary antibodies: mouse anti-TMEM40 (1:1000, Santa Cruz, CA, USA), rabbit anti-p53, rabbit anti-p21, mouse anti-CCND1, rabbit anti-c-MYC, rabbit anti-PARP1, rabbit anti-Caspase-9, rabbit anti-Caspase-3, rabbit anti-MMP1 and rabbit anti-MMP9 (1: 1000, Proteintech Group, INC, USA). The membranes were washed, incubated with appropriate HRP-conjugated secondary antibodies at room temperature, 2 h), and detected using the enhanced chemiluminescence detection system. GAPDH (1: 8000, CST, Inc, MA, USA) was used as a loading control.