Anti mmp2 antibody

Cultured cells or tissues were harvested and lysed with RIPA lysis buffer (Beyotime, China) for 30 min on ice. After centrifugation at 12,000 g for 20 min, the concentration of proteins was measured using Bradford's reagent (Bio-Rad laboratories, USA). The protein samples were denatured by boiling for 10 min and loaded onto SDS–PAGE gel for electrophoresis. The proteins were transferred onto PVDF membrane (Millipore, USA) which was then incubated in the blocking solution (5% FBS) at room temperature for 1 h. The anti-REPS2 antibody (Abcam, UK) or anti-RalBP1 antibody (Santa Cruz, USA), anti-RAC1 antibody (GeneTex, USA), anti-CDC42 antibody (GeneTex, USA), anti-MMP9 antibody (Proteintech, USA), anti-MMP2 antibody (Proteintech, USA), anti-cyclinD1 antibody (Abcam, UK), anti-EGFR antibody (Abcam, UK), anti-pEGFR antibody (Abcam, UK), anti-AKT antibody (Abcam, UK), anti-pAKT antibody (Abcam, UK), anti-Erk antibody (Abcam, UK) and anti-pErk antibody (Abcam, UK) was added into blocking solution and incubated at 4°C overnight. The membranes were subsequently incubated with HRP-labeled goat anti-rabbit IgG for 1.5 h at room temperature. Protein expression was normalized against GAPDH expression (RD, USA) or β-actin expression (Bioworld, USA). Bands were visualized by employing the BeyoECL Plus DetectionSystem (Beyotime, China) and Bio-Rad Image Lab Software (CA, USA).

For western blot analysis, total protein was extracted from MLE-12 cells and lung homogenates with M-PER and T-PER Protein Extraction Reagents (Thermo, Rockford, IL, USA), respectively which containing 0.1 mM of phenylmethyl sulfonyl fluoride (Sigma, St. Louis, MO, USA) according to manufacturer’s protocol. 20–40 µg proteins were separated by 10% SDS-PAGE under a constant voltage of 120 V for 2 h and then transferred to a nitrocellulose membrane at a constant voltage of 300 V for 1 h using a Mini-PROTEAN tetra system (Bio-Rad, Hercules, CA, USA). After blocking with phosphate buffer saline containing 0.05% Tween-20 (PBST) and 5% non-fat dry milk, the membranes were incubated at 4 °C overnight with TBST and 5% milk containing primary antibodies at following dilution ratios: anti-β-catenin antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA); anti-MMP9 antibody (1:500, Proteintech, Chicago, IL, USA); anti-MMP2 antibody (1:500, Proteintech) and anti-GAPDH antibody (1:2000, Cell Signaling Technology, Beverly, MA, USA). The membranes were then washed three times with PBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG, HRP-linked antibody (1:5000, Cell Signaling Technology) for 1 h at room temperature (RT), followed by washing with PBST. Proteins were visualized by chemiluminescence (ECL, Amersham Biosciences, GE Healthcare, UK).