Fig. 3.

Constitutively active (CA) Rho and ROCK but not Rac and PAK induce cof phosphorylation in tubular cells. A and B: LLC-PK1 cells grown on coverslips were transfected with one of the indicated constructs (1 μg DNA) encoding for the Myc epitope-tagged constitutively active form of Rho, Rac, ROCK, and PAK. Forty-eight hours later, the cells were briefly washed, fixed, and doubly stained for phosphorylated cof (red) and the Myc epitope (green) to identify the successfully transfected cells. Identical cells on the corresponding images are marked with identical symbols (arrows or asterisks). C: cells grown in 10-cm dishes were transfected with empty vector (Mock), CA-Rho, or CA-Rac and lysed 48 h later. Proteins of the whole cell lysates were separated by SDS-PAGE and subjected to Western blotting using anti-phospho-cof. The blots were reprobed with anti-Rho and anti-Myc antibodies to detect all and endogenous Rho proteins, respectively. Despite the fact that the transfection efficiency was only a few percent (< 10%), a highly significant (P < 0.001) 1.44-fold increase was detected in the phospho-cof content of the CA-Rho-transfected monolayers. This increase corresponds to a 5- to 10-fold increase in the CA-Rho expressing cells. D: cells were transfected with empty vector or Myc-tagged CA-ROCK or CA-PAK1 and then processed as in C. Tubulin was used as loading control. Note that CA-ROCK caused a highly significant increase in the phospho-cof content of the entire cell population (with a few % transfection) originating from a large increase present in the ROCK-expressing cells. CA-PAK failed to increase cof phosphorylation despite the fact that PAK1 expression was stronger than ROCK expression, as verified by the Myc signal.