Figure 7: GIV activates Gαi and suppresses the antifibrogenic cAMP/PKA/pCREB signals in HSCs.

(a) Lysates of Lx2 cells were used as the source of full-length GIV in pull-down assays with GST or GST-Gαi3 preloaded with GDP alone (inactive) or with AlF₄⁻ (active) and immobilized on glutathione beads. Bound GIV and Gβγ were analysed by IB. The GST proteins are shown with Ponceau S stain. (b) Serum-starved Lx2 cells were stimulated with a variety of ligands before lysis. Equal aliquots of lysates were subjected to immunoprecipitation with Gαi:GTP mAb, which recognizes active conformation of the G protein and immune complexes were analysed for Gαi3 by IB. (c) Lx2 cells treated with control (Scr) or GIV siRNA were transfected with siRNA-resistant GIV-WT or GIV-FA and maintained in 0.2% FBS. Lysates were analysed for active Gαi3 as in b. (d) Lx2 cells transfected with GIV-WT or GIV-FA were stimulated with PDGF-BB before lysis. Lysates were analysed for cAMP using radioimmunoassay and normalized to total protein. Results are displayed as fold change in cAMP (y axis). (e) Lx2 cells transfected with GIV-WT or GIV-FA were analysed for GIV, phospho(p)Akt, phospho(p)CREB and tubulin by IB. The ratio of pCREB/tubulin was ~2.5-fold higher in cells expressing GIV-FA than in those expressing GIV-WT, as determined using band densitometry. (f) Lx2 cells infected with GIV-WT-HA or GIV-FA-HA adenoviruses were maintained in 0.2% FBS, fixed and stained for hemagglutinin (HA) (GIV, green), phospho(p)CREB (red) and DAPI/DNA (blue) and analysed using confocal microscopy. Arrowhead=GIV-expressing cell. Star=nuclear p CREB. Scale bar=10 μm. Results display % cells with nuclear pCREB (y axis) (g). Error bars represent mean±s.d. n=3. Statistical significance was assessed with two-tailed Student’s t-test.