Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with <t>glutathione-Sepharose</t> beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.
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