Figure 5.

PhLP binds nascent Gβ in the absence of Gγ and requires S18–20 phosphorylation for Gγ to associate with the complex. (A) HEK-293 cells were transiently cotransfected with wild-type PhLP-myc in combination with Gα_i3, Flag-Gβ₁ and/or HA-Gγ₂ as indicated. After 48 h, the cells were pulsed with [³⁵S]methionine for 30 min and extracts were immunoprecipitated using antibodies to the PhLP-myc, Flag-Gβ₁ or HA-Gγ₂ epitope tags. The co-immunoprecipitating proteins were resolved on 10% Tris–Glycine–SDS gels for PhLP, Gα and Gβ and 16.5% Tris–Tricine–SDS gels for Gγ. Radioactive protein bands were detected using the phosphorimager. The gel shown is representative of similar gels from three separate experiments. (B) HEK-293 cells were transiently transfected with PhLP-myc, PhLP S18–20A, PhLP 132–135A or PhLP Δ1–75 as indicated along with Flag-Gβ₁- and HA-Gγ₂-expressing vectors. After 48 h, the cells were pulsed with [³⁵S]methionine for 30 min, extracts were immunoprecipitated and were analyzed as in panel A. Band intensities were quantified and the indicated molar ratios of binding partners were calculated as in Figure 2B. A representative gel is shown. Bar graphs represent the average molar ratios±s.e. from three separate experiments (^*P<0.01, ^**P<0.001).