FIGURE 2.

CLP36 and α-actinin-4 co-localize and form a complex in human podocytes. A, human podocytes were cultured under permissive (lane 1) or nonpermissive conditions (lane 2) for 14 days. The cell lysates (4 μg of lysates/lane) were analyzed by Western blotting with Abs recognizing CLP36 or tubulin (as a loading control). B, cell lysates were mixed with an anti-CLP36 Ab or a control Ab that does not recognize CLP36. CLP36 and control immunoprecipitates were analyzed by Western blotting with anti-CLP36 and anti-α-actinin-4 Abs. C–H, human podocytes were dually stained with mouse anti-α-actinin-4 Ab and goat anti-CLP36 Ab (C and D) or FITC-conjugated phalloidin and goat anti-CLP36 Ab (F and G). The primary mouse and goat Abs were detected with secondary Rhodamine Red-conjugated anti-mouse IgG Ab and Cy2-conjugated anti-goat IgG Ab (C and D). Goat anti-CLP36 Ab in G was detected with a Rhodamine Red-conjugated anti-goat IgG Ab. Scale bar, 10 μm. The immunofluorescent images were pseudo-colored and merged using the National Institutes of Health ImageJ program (E and H).