FIG. 6.

PLCγ1 interaction with PDZ-RhoGEF. (A) HEK 293 cells were transfected without or with FLAG-tagged PDZ-RhoGEF [(FLAG)-PRG] and Myc-tagged wild-type (WT) PLCγ1 or its indicated mutants. From cell lysates, PDZ-RhoGEF was immunoprecipitated (IP) and precipitates were then separated and immunoblotted (IB) with the indicated antibodies. (B) HEK 293 cells transfected with Myc-tagged PLCγ1 and FLAG-tagged wild-type PDZ-RhoGEF or its indicated mutants were lysed, and PLCγ1 was immunoprecipitated. Precipitates were analyzed by immunoblotting using the indicted antibodies. (C and D, left panels) HEK 293 cells were cotransfected with SRE.L firefly luciferase reporter plasmid, Renilla luciferase expression plasmid without (−) or with various PDZ-RhoGEF versions, the SH3 domains of Grb2 and PLCγ, or constitutively active Gα₁₃ (Gα₁₃QL) as indicated, and relative luciferase activity was determined as described in Materials and Methods. Shown values are the means of three independent experiments ± standard deviations. (Right panels) HEK 293 cells were transfected with wild-type PDZ-RhoGEF (WT) or its mutant (P1335A/P1338A) and were incubated without or with 150 nM Sema4D (C) or were cotransfected with control or with a constitutively active version of Gα₁₃ (Gα₁₃QL) (D). Thereafter, RhoA activity was determined as described in Materials and Methods section. ***, P ≤ 0.001.