Figure 3.

Claudin-2 silencing activates RhoA. A–D, WT LLC-PK₁ cells (A and D) or MDCK cells (B) or HA-claudin-2 expressing LLC-PK₁ cells (C) were transfected with NR or Cldn-1, -2, or -7–specific siRNAs, as indicated. 48 h after transfection, the cells were lysed, and active RhoA was captured using GST–RBD-coupled beads. Precipitated and total RhoA were detected by Western blotting. RhoA in the precipitate (active) is indicated by the arrowhead. Note that in some blots, the antibody also visualized a nonspecific band above the RhoA-specific band. The identity of the RhoA band was verified by silencing. Active RhoA was normalized to the corresponding input (total) RhoA and in each experiment expressed as fold from NR siRNA transfected control. The indicated claudins were detected in total cell lysates to verify silencing. Graphs show means ± S.E. n = 3–5. E, active RhoA staining is elevated in Cldn-2 KO mice. Active RhoA was detected in kidney sections obtained from WT or Cldn-2 KO mice using a RhoA-GTP–specific antibody (left top row). The right top row shows labeling with the secondary antibody alone. Middle row, nuclear stain with DAPI; bottom row, merged images. Bar, 50 μm. Intensity of the labeling was quantified using the Zen software in regions of 100-μm diameter (>30 regions/animals in three KO and two WT animals). Average labeling in samples exposed to the secondary antibody only was subtracted from each intensity value (means ± S.D.)