Figure 2

TRPV5 protein detected by immunoblot. Incubation of primary cultures of SCCD for 24 h in the absence and presence of 100 nM 1,25- (OH)₂vitamin D₃. Protein expression of TRPV5 was compared with β-actin. A) Cultured SCCD cells with or without 1,25-(OH)₂vitamin D₃; kidney cortex and papilla were used for positive and negative controls. The blot was stripped of TRPV5 antibody and re-probed for beta-actin. B) Antigenic peptide blocked 80 kDa band. A band at 42 kDa is β-actin. The blot was directly probed for beta-actin without stripping since the TRPV5 antibody did not produce bands at the molecular weight of actin. C) PNGase F did not affect any TRPV5 bands. A band at 36 kDa is PNGase F itself. D) Intensity of 80 kDa TRPV5 bands standardized to β-actin. The mean intensity in 1,25-(OH)₂vitamin D₃ treated cells was larger but not statistically significant. Ctrl, control; Vit.D, 1,25-(OH)₂vitamin D₃ ; ns, P > 0.05; n = 14 each condition.