Figure 2.

CXCR4 regulates the arrestin-2/STAM-1 interaction. (A) HeLa cells transiently transfected with HA-arrestin-2 were serum starved as described in Materials and Methods, followed by treatment with 30 nM CXCL12 for 30 and 60 min. Cell lysates were subject to immunoprecipitation using monoclonal anti-HA and isotype control antibodies. Immunoprecipitates and lysates were analyzed by SDS-PAGE and immunoblotting to detect endogenous STAM-1 and HA-arrestin-2. Immunoblots were subject to densitometric analysis, and the bar graph represents the average STAM-1 binding ± SEM normalized to the level of HA-arrestin-2 in the immunoprecipitates. STAM-1 binding to arrestin-2 was significantly increased upon agonist treatment as compared with vehicle. Data were analyzed by one-way ANOVA followed by a Bonferroni's post hoc test (*p < 0.05). (B) STAM-1 is preferentially ubiquitinated upon CXCR4 activation. HEK293 cells cotransfected with HA-CXCR4, FLAG-STAM-1, or FLAG-STAM-2 and HA-ubiquitin were treated with 100 nM CXCL12 for 30 min. FLAG-STAM-1/2 were immunoprecipitated using an anti-FLAG antibody, followed by SDS-PAGE and immunoblotting to detect incorporated HA-ubiquitin. Blots were stripped and reprobed for FLAG-STAM-1/2 to assess loading. Cell lysates were analyzed for the presence of HA-CXCR4. Shown are representative blots from one of three independent experiments.