WB using anti-Pdcd4 pS457 antibody shows detection of phosphorylated Pdcd4 (indicated by arrowhead at ~62 kDa) in NIH-3T3 cells after 5 min treatment with 30 ng/mL PDGF (lane 2). No reactivity is seen for unstimulated (non-phosphorylated) NIH 3T3 cells (lane 1). The membrane was probed with the primary antibody at a 1:2,000 dilution, overnight at 4?. For detection HRP conjugated Rb-a-Mouse IgG was used at a 1:20,000 dilution in blocking buffer for 1 h at 4?.