Rabbit anti mmp2

Total proteins were extracted using RIPA buffer (with phenylmethylsulfonyl fluoride at 100:1). After the proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis, they were transferred onto polyvinylidene difluoride membranes. Subsequently, the membranes were incubated with 5% bovine serum albumin (Solarbio, Beijing, China) for 2 h at room temperature (22–25 ℃), followed by incubation with rabbit anti-TAGLN2 antibody (1:1000), rabbit anti-E-cadherin (1:2000), rabbit anti-N-cadherin (1:2000), rabbit anti-vimentin (1:2000), rabbit anti-MMP2 (1:800), and rabbit anti-GAPDH (1:8000) (all purchased from ProteinTech Group Inc., Wuhan, China) at 4 °C for 14–16 h. Thereafter, the collected membranes were washed with TBST (Tris-buffered saline, 0.1% Tween 20) three times. Horseradish peroxidase-labeled goat anti-rabbit antibody (1:2500; ProteinTech Group Inc.) or horseradish peroxidase-labeled goat anti-mouse antibody (1:10,000; Proteintech Group Inc.) was used as a secondary antibody, and after incubation at room temperature (22–25 ℃) for 2 h, the membranes were washed with TBST three times and detected via enhanced chemiluminescence.

Whole cell extracts were lysed in RIPA Lysis buffer (Beyotime, P0013B) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Then protein concentration of lysates was determined by BCA protein concentration determination kit (Beyotime, P0010). Cell lysates containing equal amount protein were resolved on a 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, IPVH00010). After separate incubation with rabbit anti-p65 (CST, #8242), rabbit anti-p-p65 (CST, #3033), rabbit anti-MMP2 (proteintech, 10,373–2-AP), rabbit anti-MMP9 (proteintech, 10,375–2-AP), rabbit anti-E-cadherin (proteintech, 20,874–1-AP), rabbit anti-IKBα (abcam, Ab32518), rabbit anti-ICAM-1 (proteintech, 10,831–1-AP), rabbit anti-VCAM-1 (Affinity, DF6082), rabbit anti-E-selectin (proteintech, 20,894–1-AP), rabbit anti-GAPDH, mouse anti-P-selectin (proteintech, 60,322–1-Ig), mouse anti-MCP-1 (Affinity, BF0678), followed by horseradish peroxidase-conjugated secondary antibody, the membranes were visualized by ECL chemiluminescence.

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.