Mmp 2 antibody

First, the whole protein was extracted by lysing cervical cancer cells with RIPA buffer before measuring the protein concentration via a BCA protein assay kit (A8020‐5, Roche, Basel, Switzerland). The cells were dissolved with SDS‐PAGE loading buffer and transferred into PVDF membranes. Then, the cells were incubated with diluted primary antibodies at 4°C after blocking with 5% skim milk dissolved in TBST for 1 hour. Primary antibodies used include HSDL2 antibody, E‐cadherin antibody, Vimentin antibody, Snail antibody, Slug antibody (Cell Signaling Technology, Boston, USA), MMP‐2 antibody (Affinity, Cincinnati, USA), FASN antibody, ACSL1 antibody, SREBP1 antibody (Proteintech) and GAPDH antibody (CWBIO). Next, goat antimouse IgG conjugated to HRP was applied as secondary antibody for 1 hour at room temperature. The cells were shortly incubated by ECL detection reagent, then detected by Imager.

GI-LI-N and SK-N-SH cells were harvested and lysed with RIPA cell lysis buffer (Thermo Fisher Scientific) with protease inhibitors. The protein concentration was measured by bicinchoninic acid method (Thermo Fisher Scientific). Then the extracted protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) on ice. Next, after being blocked for 1 h with 5% non-fat milk at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C. MMP-2 antibody (1:2,000), E-cadherin antibody (1:2,000), N-cadherin antibody (1:1,000), vimentin antibody (1:1,000), phosphatidylinositol 3-kinase (PI3K) (#4,257, 1:1,000), phosphorylated (p)-PI3K (#17,366, 1:1,000), serine/threonine kinase 1 (AKT) (#9,272, 1:1,000), p-AKT (#9,611, 1:1,000), and GAPDH antibody (1:1,000) were purchased from Affinity Biosciences (Changzhou, China). Moreover, membranes were incubated with the corresponding HRP-conjugated second antibodies HRP Goat Anti-Rabbit IgG (H+L) (1: 5000, ABclonal, Wuhan, china) or HRP Goat Anti-Mouse IgG (H+L) (1: 5000, ABclonal), and protein signals were visualized with enhanced chemiluminescence (Millipore).