Rabbit anti smad2 antibody

The details of the experimental procedures were described previously.^{35 (link)} The antibodies used in this assay were the following: mouse anti-ZNF32 antibody (1:200), mouse anti-TGF-βR2 antibody (1:100), rabbit anti-β-actin antibody (1:800, Santa Cruz Biotechnology), rabbit anti-ERK antibody (1:1000, Cell Signaling Technology, Lexington, KY, USA), rabbit anti-p-ERK antibody (1:500, Cell Signaling Technology), rabbit anti-SMAD2 antibody (1:500, Cell Signaling Technology), rabbit anti-pSMAD2 antibody (1:200, Cell Signaling Technology), rabbit anti-MEK antibody (1:1000, Cell Signaling Technology), rabbit anti-p-MEK antibody (1:500, Cell Signaling Technology), horseradish peroxidase-conjugated secondary antibody to rabbit IgG (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish peroxidase-conjugated secondary antibody to mouse IgG (1:8000, Santa Cruz Biotechnology).

Cells were lysed with RIPA buffer (Millipore, Billerica, MA, United States) that contained phosphatase and proteinase inhibitors. The protein concentration of the lysates was measured by the Bradford assay (Bio-Rad, Hercules, CA, United States). Aliquots of lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blot analysis. Primary antibodies included a rabbit anti-TGFBR1 antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-beta actin antibody (1:10,000, Sigma-Aldrich), rabbit anti-phospho-Smad2 antibody (1:1,000, Millipore), and rabbit anti-Smad2 antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States). Secondary antibodies were a horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (1:10,000, GE Healthcare, Piscataway, NJ, United States) and HRP-linked anti-rabbit IgG antibody (1:10,000, GE Healthcare). Immunoreactive proteins were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, United States) with an ImageQuant LAS4,000 (GE Healthcare).