Ab180733

Total protein was extracted from the transfected HL-7702 cells using RIPA buffer. The protein concentration was then quantified and normalized to ensure equal loading into polyacrylamide gels. Equal amounts of protein were subjected to 10% polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membrane was incubated with the antibody of PROC (1:1000, ab180733, Abcam, UK), cyclooxygenase-2 (COX-2) (1:1000, ab179800, Abcam, UK), vascular endothelial-derived growth factor (VEGF) (1:2000, ab46154, Abcam, UK), B cell lymphoma-2 (Bcl-2) (1:1000, ab182858, Abcam, UK) and Bcl-2 associated X (BAX) (1:800, ab32503, Abcam, UK) overnight. The β-actin antibody (Cell Signaling Technology,USA) was served as the internal control. The next day, the membrane was incubated with the secondary antibody. The bands were visualized using an ECL luminescent reagent (Millipore, USA).

Protein isolation was done using radioimmunoprecipitation assay buffer (CWBio, Beijing, China), and protein concentration was detected using a bicinchoninic acid protein assay kit (Tiangen, Beijing, China). Then, an equal amount of proteins was split by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% nonfat milk for 1 h at indoor temperature. Next, the membranes were immunoblotted with primary antibodies against GAPDH (ab181602; Abcam), total p65 (t-p65; ab16502; Abcam), phosphorylated p65 (p-p65; ab86299; Abcam), B-cell lymphoma-2 (Bcl-2, ab196495; Abcam), BCL2-associated X (Bax, ab180733; Abcam), cleaved-caspase 3 (C-caspase 3, ab49822; Abcam), or total-caspase 3 (t-caspase 3, ab90437; Abcam) overnight at 4°C and then incubated with corresponding secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. Finally, the protein bands were exposed through an enhanced chemiluminescence reagent (Vazyme) and analyzed by software Image J.