WB using Anti-SMAD1 pS206 antibody shows detection of endogenous phosphorylated SMAD1 in whole cell lysates from HEPG2 (lanes 1-4) derived cell lines treated with PBS, BMP2 (5ng/mL), EGF (1ng/mL), or NaCl for 1h at 37°C before harvest. Primary antibody was used at 1:500 and pre-incubated before reacting with blot as follows: top row - with PBS, middle row - with the immunizing phosphorylated peptide and bottom row - with control or non-phosphorylated peptide. HRP-conjugated-Rabbit IgG was 1:3,000.