FIGURE 1.

A siRNA library screen reveals an important role for Rab11B in regulation of PAR1 surface expression. A and B, HeLa cells stably expressing FLAG-tagged PAR1 were transfected with ns, μ2 adaptin subunit siRNA, or SMARTpool siRNAs targeting 140 distinct membrane trafficking proteins. After transfection, cells were either left untreated (Control) or treated with 100 μm SFLLRN (PAR1 agonist peptide) for 15 min at 37 °C. Cells were fixed, and the amount of PAR1 remaining on the cell surface was quantified by ELISA. The data (mean, n = 2) are representative of two independent experiments performed in duplicate and are expressed as the percent of ns siRNA control. C, PAR1-expressing HeLa cells were transfected with ns, μ2 adaptin, Rab11B #6, #7, and #8 siRNAs, or the Rab11B SMARTpool (sp) siRNAs, and PAR1 cell surface expression was determined by ELISA. Data (mean ± S.D., n = 3) are expressed as the percent of ns siRNA control. The inset is a representative immunoblot (IB) of cell lysates from similar siRNA transfections probed with anti-Rab11B or anti-actin antibodies.