Rabbit anti human mmp 2

The cells were lysed in RIPA buffer (Heart Biological Technology Co., Ltd., Xi'an, China) containing 1% aprotinin, 1% activated Na₃VO₄ and 1% PMSF on ice. The protein concentration was detected according to the instructions of the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins (50 µg) were loaded onto a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated by electrophoresis and transfered onto PVDF membranes (Millipore, Boston, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with rabbit anti-human PKCζ (1:1,000, #sc-216; Santa Cruz Biotechnology, Inc., CA, USA), rabbit anti-human MMP2 (1:1,000, #10373-2-AP), MMP9 (1:500, #10375-2-AP), mouse anti-human β-actin (1:1,000, #60008-1-lg) (both from China Branch of Proteintech Co., Hubei, China) and rabbit anti-human phospho-PKCζ antibodies (1:500, #9378; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. The following day, the membranes were sequentially incubated with HRP-conjugated goat anti-rabbit IgG (1:3,000, #A0208) or HRP-conjugated goat anti-mouse IgG (1:3,000, #A0216) (Beyotime, Shanghai, China) at 37°C for 1 h. Immunoreactive proteins were detected using ECL reagent (Millipore) and the FluorChem FC system (Alpha Innotech, San Leandro, CA, USA). Bands were quantitated using ImageJ software.

The supernatant from whole-cell lysates was harvested and immunoprecipitated using the indicated primary antibodies or non-specific IgG as a negative control, which was precleared by protein G magnetic beads at 4°C overnight. Proteins were separated on 10 or 12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): mouse anti-human Gal-1, rabbit anti-human H-Ras, rabbit anti-human ERK, mouse anti-human p-ERK, rabbit anti-human c-Jun, mouse anti-human MMP-9, rabbit anti-human p21, rabbit anti-human Bcl-2, and rabbit anti-human Raf-1 antibodies. Rabbit anti-human p-AKT (Ser473), rabbit anti-human p-Raf-1 (Ser338), and rabbit anti-human MMP-2 antibodies were purchased from Cell Signaling (Danvers, MA, USA). The mouse anti-GAPDH antibody was purchased from KangChen Bio-tech (Shanghai, China). All experiments were repeated at least twice.