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. 2014 Feb 19;289(14):9502–9518. doi: 10.1074/jbc.M113.505743

FIGURE 1.

FIGURE 1.

TAT-Neph1-CD is transduced in podocytes and remains functional. A, recombinantly expressed TAT-Neph1CD was purified, and its purity and identity were assessed by staining with Coomassie and Western blotting with Neph1 antibody, respectively. B, TAT-Neph1CD protein (5 μg/ml) was transduced in cultured podocytes for the indicated time periods ranging from 30 min to 24 h and subjected to Western blotting (WB) with Neph1 antibody. The protein rapidly transduced inside the cells within 30 min and was detected even at 24 h post-transduction. C, in a similar fashion, TAT-GFP was transduced for indicated times and was used as a control. D, TAT-Neph1CD was transduced in cultured human podocytes for a period of 30 min, washed, and fixed with 4% PFA (1× PBS). His primary antibody and Alexa Fluor 594 secondary antibodies were used to label the transduced TAT-Neph1CD (arrows), and the immunofluorescence imaging analysis was performed using a fluorescent microscope (×60 magnification). Additionally, the cells were visualized by differential interference contrast (DIC) microscopy. E, TAT-Neph1CD protein was labeled with Texas red dye and transduced in cultured human podocytes for 30 min. The cells were then washed and fixed with 4% PFA (in 1× PBS) and analyzed by immunofluorescence imaging that showed significant accumulation of fluorescent TAT-Neph1CD inside the transduced podocytes as compared with the untransduced control podocytes. F, statistical analysis of the TAT-Neph1CD transduction from D suggested that 94.6 ± 1.5% of the podocytes were transduced with this protein. G, statistical analysis of the Texas red TAT-Neph1CD transduction suggested that 96.3 ± 1.5% of podocytes were transduced with this protein. H, TUNEL assay was performed on transduced and untransduced podocytes. No apoptosis could be seen in the podocytes that were transduced with 10 times (50 μg/ml) higher than the usual dose (5 μg/ml) of TAT-Neph1CD in podocytes. Bright field and DAPI images were also obtained to visualize the overall cell population. I, ability of transduced TAT-Neph1CD to interact with endogenous ZO-1 was examined in a pulldown experiment using Ni-NTA beads. Podocytes transduced with TAT-Neph1CD were lysed, and the Neph1-ZO-1 complex was pulled down using nickel beads, separated on SDS-PAGE, and analyzed by Western blotting to detect the presence of ZO-1 in the complex. Scale bars, 10 μm (D), 20 μm (E); 20 μm (H).