PAR1 expression is differentially regulated by Rab11A and Rab11B.
A and B, HeLa cells stably expressing FLAG-PAR1 or FLAG-PAR1 0K mutant were transfected ns, Rab11A, or Rab11B siRNAs for 72 h. A and D, HeLa cells were fixed, and PAR1 wild type and 0K mutant surface expression was quantified by ELISA. Data (mean ± S.D., n = 3) are representative of three independent experiments, expressed as the percent of ns siRNA control, and were analyzed by ANOVA (*, p < 0.05; **, p < 0.01; ***, p < 0.001). B and E, HeLa cell lysates were immunoblotted (IB) for PAR1 wild type and 0K mutant protein expression using anti-FLAG antibodies. Data (mean ± S.D., n = 3) are representative of three separate experiments, expressed as the percent of ns siRNA control, and were analyzed by ANOVA (*, p < 0.05; **, p < 0.01). Cell lysates were also probed with anti-Rab11A, -Rab11B, and -GAPDH antibodies as controls. C, HeLa cells were fixed, permeabilized, and immunostained with anti-FLAG antibody to detect PAR1 wild type and 0K mutant expression. Similar results were observed in three different experiments. Scale bar, 10 μm.