Characterization of the epitope, specificity, and sensitivity of the RGS14 mouse monoclonal antibody. A: Cartoons depicting the domain structure of Flag-tagged RGS14 truncation mutants used to map the region recognized by the antibody. RGS, Regulator of G protein signaling domain; RBD, Ras/Rap- binding domain; GPR, G protein regulatory motif. B: HEK293 cell lysates transfected with Flag-tagged RGS14 or truncation mutant cDNAs were subjected to SDS-PAGE and immunoblotting with anti-RGS14 antibody (1:5,000). Equal amounts of these samples were separately subjected to SDS-PAGE and immunoblotting with an anti-Flag antibody (1:25,000) to verify cDNA expression. C: Wild-type mouse brain homogenate (Lane 1) and HEK293 cell lysates transfected with Flag-RGS14 (Lane 2), GFP-RGS12 TS (Lane 3), Flag-RGS10 (Lane 4), RGS2-HA (Lane 5), RGS4-HA (Lane 6), RGS16-HA (Lane 7), or empty vector (Lane 8, pCDNA3.1) were subjected to SDS-PAGE and immunoblotting with the anti-RGS14 antibody (1:5,000). Equal amounts of these samples were separately subjected to SDS-PAGE and immunoblotting with either an anti-Flag antibody (Lanes 2 and 4, 1:25,000), anti-GFP antibody (Lane 3, 1:1,000), or anti-HA antibody (Lanes 5–8, 1:1,000) to confirm protein expression. GFP, green fluorescent protein; HA, hemagglutinin. D: A serial dilution of purified thioredoxin-and hexa-histidine-tagged RGS14 (TxH6-RGS14) at 100 ng, 50, 25, 12.5, 6.25, 3.12, 1.50, 0.75 ng was subjected to SDS-PAGE and immunoblotting with the anti-RGS14 antibody (1:5,000).