Figure 3.

Orthologues of GPR35 promote activation of Gα₁₃. Studies with a Gα₁₃ antibody that interacts selectively with GTP-Gα₁₃. Cells harbouring either FLAG-hGPR35-eYFP (A, B) or FLAG-rGPR35-eYFP (C, D) were either untreated (- dox) or treated (+ dox) with doxycycline (concentrations as noted). Cells were then transfected with either wild type Gα₁₃ or a constitutively active Gln²²⁶Leu Gα₁₃ variant (CAM). Cells were treated with or without 100 µM zaprinast as noted for 30 min. Samples were immunoprecipitated with an active-state Gα₁₃ antibody and these samples (GTP-Gα₁₃) and cell lysates (Total Gα₁₃) were resolved by SDS-PAGE and subsequently immunoblotted with a polyclonal Gα₁₃ antiserum. In (B and D) the relative amount of GTP-Gα₁₃ and total levels of Gα₁₃ were assessed by analysis of such immunoblots following different periods of exposure to 100 µM zaprinast (means ± SEM, n = 3). (E) Transfection with either wild-type or CAM Gα₁₃ results in a substantial enhancement of expression. Cells induced to express FLAG-hGPR35-eYFP were transfected transiently with either wild-type Gα₁₃ or the CAM variant. An empty vector transfection (-Gα₁₃) was also performed. Lysates from cells were resolved by SDS-PAGE and immunoblotted with the polyclonal Gα₁₃ antiserum.