Exogenously expressed SAR1 binds to Giα2, 3 proteins to activate Jun and increase γ-globin chain in K562 and CD34⁺ cells. (A) SAR1- or vector control–stably transfected K562 cells were subjected to Giα activation assay. β-actin was used as a loading control. (B) SAR1- or vector only–stably transfected K562 cells were transfected with control, Giα1, Giα2, Giα3, Gαq, or Gα11 siRNA at day 0 and day 1, then collected at day 3 for western blot analysis to examine phospho- and total c-Jun protein expression (left panel) and real-time PCR to analyze γ-globin mRNA expression (right panel). Mock-transfected cells were used as a negative control. β-actin was used as a loading control. Fold increase was calculated relative to expression in corresponding control siRNA-transfected cells after normalization with β-actin gene expression. *P < .001 vs SAR1-overexpressed K562 cells transfected with control siRNA. Error bars represent SD of the mean of 3 independent experiments. (C) CD34⁺ cells were cotransfected with SAR1-expressing or empty vector and scrambled siRNA or Giα1, Giα2, or Giα3 siRNA at day 5 of differentiation, then collected at day 7 for western blot analysis to examine phospho- and total c-Jun protein expression (left panel) and real-time PCR to analyze γ-globin chain mRNA expression (right panel). Fold increase was calculated relative to expression in corresponding control siRNA-transfected cells after normalization with β-actin gene expression. *P < .001 vs CD34⁺ cells transfected with SAR1 and control siRNA. Error bars represent SD of the mean of 3 independent experiments. (D) Whole-cell lysates from SAR1-V5– or vector control–stably transfected K562 cells were immunoprecipitated with normal IgG, anti-Giα1, -Giα2, -Giα3, -Gαq, or -Gβ antibody, then the immunoprecipitates were subjected to western blot analysis with anti-V5 antibody. β-actin was used as a loading control. (E) SAR1-V5–stably transfected K562 cells were transfected with vector only, Giα1-Flag, Giα2-Flag, Giα3-Flag, or Gαq-Flag. Whole-cell lysates were immunoprecipitated with anti-Flag or anti-V5 tag antibody, and the immunoprecipitates subjected to western blot analysis with anti-V5 or anti-Flag antibody, respectively.