Peroxidase conjugated anti rabbit immunoglobulins

Cells were lysed in an SDS sample buffer containing 0.5 M Tris–HCl, 10% SDS, glycerol, bromophenol blue, and 3% 2-mercaptoethanol. They were boiled at 95 °C for 5 min, and then the protein was fractionated on 7.5–12.5% SDS-polyacrylamide gel electrophoresis (PAGE) (e-PAGEL, ATTO Corporation, Japan). The protein was transferred to PVDF membranes (Immobilon-P Transfer Membrane): the membranes were blocked for 1 h at room temperature to block the non-specific binding of the protein and incubated with primary antibodies at 4 °C overnight. The primary antibodies were as follows: anti-sema3G antibody (Sigma Aldrich, HPA001761, 1:1000 dilution), anti-phospho-p65 antibody (Santa Cruz, sc-101749,1:1000 dilution), anti-p65 antibody (Santa Cruz, sc-372, 1:1000 dilution), anti-phospho-ERK antibody (Cell Signaling, #9106, 1:1000 dilution), and anti-ERK antibody (Cell Signaling, #9102, 1:1000 dilution). The blots were then washed and incubated with second antibodies, peroxidase-conjugated anti-rabbit immunoglobulins (1:2500 dilution, GE healthcare), or goat anti-mouse IgG-HRP (1:2500 dilution, Santa Cruz: sc-2055) at room temperature for 1 h. After washing several times, the antibody binding sites were visualized using an ECL Western blotting detection system (GE healthcare: RPN2106). The blots were quantified using a ChemiDoc MP ImageLab PC system (BIO-RAD).

Podocytes were lysed in a SDS sample buffer containing 0.5 M Tris-HCl, 10% SDS, glycerol, bromophenol blue, and 3% 2-mercaptoethanol. They were boiled at 95°C for 10 min, and then the protein was fractionated on 10%–15% polyacrylamide gels (e-PAGEL, ATTO Corporation, Japan). The protein was transferred to PVDF membranes (Immobilon-P Transfer Membrane); the membranes were blocked for 1 h at room temperature to block the nonspecific binding of the protein and incubated for 18 h at 4°C with primary antibodies. The primary antibodies that were used were as follows: anti-VPAC1 antibody (1 : 200 dilution, Santa Cruz: sc-30019), anti-phospho-p44/42 MAPK (extracellular signal-regulated kinase (ERK1/2)) antibody (1 : 1000 dilution, Cell-Signaling: number 9106), and anti-p44/42 MAPK (ERK1/2) antibody (1 : 1000 dilution, Cell-Signaling: number 9102). The blots were then washed and incubated with second antibodies, peroxidase-conjugated anti-rabbit immunoglobulins (1 : 2500 dilution, GE healthcare), or goat anti-mouse IgG-HRP (1 : 2500 dilution, Santa Cruz: sc-2055) for 1 h at room temperature. After washing for several times, the antibody binding sites were visualized using an ECL western blotting detection system (GE healthcare: RPN2106). The blots were quantified using a ChemiDoc MP ImageLab PCsystem (BIO-RAD).