Figure 5.

ALIX is required for ARRDC3-mediated degradation of activated PAR1. MDA-MB-231 HA-ARRDC3 pSLIK cells were transfected with 25 nm nonspecific (NS) or 25 nm of ALIX siRNA using a combination of 12.5 nm ALIX 1 and 12.5 nm ALIX 3 siRNAs and then incubated with or without 1 μg/ml DOX for 48 h. Cells were then stimulated with 10 nm α-thrombin for the indicated times, lysed, and immunoprecipitated using the anti-PAR1 WEDE antibody. Immunoprecipitates were immunoblotted with anti-PAR1 rabbit antibody to detect PAR1 expression. ALIX, HA-ARRDC3, and β-actin expression were determined by immunoblotting cell lysates. The data (mean ± S.D. (error bars), n = 3) are represented as the fraction of PAR1 remaining relative to 0 min control and are representative of three independent experiments. Statistical significance was determined by unpaired t test (*, p < 0.05; n = 3).