FIG. 3.

Gα₁₂ and GPCRs that activate Gα₁₂ stimulate the expression and transactivation of PDGFRα. (A) Gα₁₂QL stimulates the expression and activation of PDGFRα. Lysates (500 μg) from Gα₁₂QL-NIH 3T3 cells along with vector control were immunoprecipitated with antibodies to PDGFRα and subjected to immunoblot analysis with antibodies to P-Tyr. After stripping, the blot was sequentially reprobed with PDGFRα and GAPDH. (B) Gα₁₂QL does not stimulate the expression of PDGFRβ. Lysates (500 μg) from Gα₁₂QL-NIH 3T3 cells along with vector control were subjected to immunoprecipitation with antibodies to PDGFRβ, followed by immunoblot analysis with antibodies to P-Tyr. The blot was stripped and reprobed with PDGFRβ and GAPDH. (C) Transient expression of Gα₁₂QL stimulates the expression and transactivation of PDGFRα in NIH 3T3 cells. NIH 3T3 cells were transfected with the expression vector pcDNA3 or pcDNA3 encoding Gα₁₂QL (8 μg) using Lipofectamine Plus reagent. After 24 h, the lysates (100 μg) from the transfectants were subjected to immunoprecipitation using antibodies to PDGFRα, following which an immunoblot analysis was carried out with antibodies to P-Tyr. The blot was stripped and reprobed with antibodies to PDGFRα. The blot was further probed with antibodies to Gα₁₂ and GAPDH to monitor Gα₁₂QL-expression and equal loading, respectively. (D) Transient expression of Gα₁₂QL stimulates the expression and transactivation of PDGFRα in 1321N1 astrocytoma cells. 1321N1 astrocytoma cells were transfected with vectors encoding Gα₁₂QL or pcDNA3 vector (5 μg) using Fugene 6 reagent. After 24 h, the lysates (100 μg) from the transfectants were subjected to immunoprecipitation using antibodies to PDGFRα, following which an immunoblot analysis was carried out with antibodies to P-Tyr. The blot was stripped and reprobed with antibodies to PDGFRα. The blot was stripped and reprobed with antibodies to Gα₁₂ and GAPDH to monitor Gα₁₂QL-expression and equal loading, respectively. (E) GPCR ligands, LPA and TRAP, stimulate the expression and transactivation of PDGFRα in 1321N1 astrocytoma cells. 1321N1 astrocytoma cells were serum starved for 24 h. After starvation, the cells were stimulated with 20 μM LPA or 5 μM TRAP for 6 h. The cell lysates (100 μg) were subjected to immunoblot analysis with antibodies to P-Tyr. The blot was further reprobed with antibodies to GAPDH to monitor equal loading of proteins. IP, immunoprecipitate; IB, immunoblot.