Fig. 2.

aPKC is required for the cell-surface localization of SD components, including nephrin. (A) Isolated rat glomeruli were treated with 10 µM aPKC pseudosubstrate (PS) or SC for 30 min at 37°C in HBSS(+), then subjected to the cell-surface biotinylation assay. (B) Quantification of the results in (A). (C) HCT116-nephrin cells were treated with 20 µM of aPKC-PS or SC for 2 h at 37°C and subjected to the cell-surface biotinylation assay. (D) Quantification of the results in (C). (E) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubated for 48 h and then subjected to the cell-surface biotinylation assay. (F) Quantification of the results in (E). (G) HCT116-nephrin cells were transiently transfected with aPKCλ/ζ siRNA and incubated for 70 h. Both isotypes of aPKC are expressed in HCT116 cells (data not shown). After incubation, the cells were subjected to the cell-surface biotinylation assay. (H) Quantification of the results in (G). The values shown in B, D, F and H were normalized to the appropriate control and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t-test.