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Published Pictures for 26902 Gα13-GTP mAb

13 activation assay 

13 activation assays were performed using a Gα13 activation assay kit (NewEast Biosciences) according to the manufacturer’s instructions. Flp-In™ T-REx™ 293 cells able to express FLAG-tagged human GPR35-eYFP or FLAG-tagged rat GPR35-eYFP in response to doxycycline were induced and incubated for varying times with either vehicle or agonist. Cells were immediately washed with ice-cold phosphate-buffered saline and prepared in ice-cold lysis buffer. Lysates were incubated with the anti-active-state Gα13 mouse monoclonal antibody. Gα13-GTP was then recovered using protein A/G agarose and the precipitated Gα13-GTP was detected following SDS-PAGE by immunoblot analysis using a conformation insensitive anti-Gα13 rabbit polyclonal antiserum. Parallel immunoblots of cell lysates with the polyclonal anti-Gα13 antiserum assessed total cell levels of this G protein.

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.

Endogenous Gα13 activation assay

A Gα13 activation assay was performed using a Gα13 activation assay kit. Following the instructions of the kit, cells cultured in 60-mm dishes were treated with or without drugs and lysed with 1 ml of ice-cold 1× assay buffer for 30 min. Cells were scraped into a 1.5-ml tube and centrifuged at 4°C (12,000 rpm, 10 min). The supernatants were transferred into new tubes. For negative and positive controls, GDP (1 mM) and GTPγS (100 μM) were added, respectively, and incubated at 30°C for 90 min with agitation. Then, 1 μl of anti–active Gα13 (Gα13-GTP) monoclonal antibody and 20 μl of resuspended protein A/G beads slurry were added to each tube. The mix was incubated at 4°C for 2 hours with gentle agitation and precipitated by centrifugation for 1 min at 5000g. The beads were washed three times with 0.5 ml of 1× lysis buffer and resuspended with 20 μl of 2× reducing SDS-PAGE sample buffer. The samples were boiled for 10 min at 100°C before detection by Western blotting.

Fig. 3.

Activation of the GABAB receptor leads to G13 protein activation.
(A) Illustration of the BRET-based G protein sensor. Upon receptor activation, rearrangement of the Gα subunit from Gβγ causes the BRET signal to decrease. (B) BRET experiment for Gα13 and Gαo activation analysis in HEK293 cells transfected with GABAB receptor and G protein sensor. Data are means ± SEM of three independent experiments. (C) Dose-response curve of GABA-induced BRET changes in the Gα13 and Gα1 sensors. Data are means ± SEM of three independent experiments. (D and E) Gα13-GTP pull-down analysis of the endogenous Gα13 activation. Blots are representative of four independent experiments. (F) Co–immunoprecipitation (IP) analysis of GABAB receptor and Gα13 and Gα1 interaction. Blots are representative of at least five independent experiments. All data are means ± SEM, analyzed with one-way ordinary ANOVA followed by Sidak’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the basal level.

Immunoblot analysis

Immunoblot analyses were performed according to previously published methods ( 22 ). Briefly, the cells were lysed in buffer containing 10 mM Tris–HCl (pH 7.1), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM dithiothreitol and 0.5 mM phenylmethylsulfonyl fluoride, supplemented with a protease inhibitor cocktail (Calbiochem). Proteins of interest were visualized by the ECL chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK).


Changes in the cytotoxic effect of bortezomib (BZ) by Gα 12 or Gα 13 activity modulations.

A ) The cytotoxicity of bortezomib (10 μM, 24 h treatment) was assessed in Huh7 cells that had been stably transfected with a constitutively active mutant of Gα 12 or Gα 13 (Gα 12 QL or Gα 13 QL). Data represent the mean ± SE of three independent experiments (* P < 0.05, ** P < 0.01, significant compared with wild-type (WT) cells treated with bortezomib at respective concentrations). Immunoblottings for Gα 12 or Gα 13 verified transfection efficiency. ( B ) The cytotoxicity of bortezomib was measured in SK-Hep1 cells that had been transfected with Gα 12 or Gα 13 minigene (CT12 or CT13). Data represent the mean ± SE of three independent experiments (* P < 0.05, ** P < 0.01, significant compared with wild-type cells treated with bortezomib at respective concentrations). Immunoblottings for active Gα 13 or active Rho confirmed transfection efficiency of the minigenes. Equal loading of proteins was confirmed by immunoblottings for β-actin. Results were confirmed by three independent experiments.