Analysis of O -GlcNAcylation level of cytoskeletal proteins by immunoprecipitation and immunoblotting. Total lysates of nondiabetic and diabetic kidneys were immunoprecipitated with anti-actin (A), anti-α-actinin 4 (B), anti-α-tubulin antibody (C) or anti-myosin (D). The immunocomplexes were separated by SDS-PAGE, transferred to PVDF membranes, and probed with O-GlcNAc antibody (a, upper panel). The membrane was then stripped and reprobed with the antibody used for immunoprecipitation (a, lower panel). Results shown are representative of 3 independent experiments. Intensity of bands recognized by the antibody used for immunoprecipitation was quantified by scanning densitometry (b). Relative intensities of the O-GlcNAc reactive bands to bands reactive with each antibody used for immunoprecipitation were then determined (c). Data are the means ± SEM from 3 different rats. (□), Wistar rats; (■), GK rats. *P < 0.05 and **p < 0.01 vs. control Wistar rat.