Fig. 2.

Homologous agonist-mediated phosphorylation of FFA4-L. (A) Immunoblotting with isoform-specific GRK antibodies was used to verify siRNA-mediated knockdown of each in HEK293 cells. SCR is the scrambled control siRNA sequence that does not target any known human sequence. β-actin is used as a loading control for each set. Representative immunoblots are shown (upper) and pooled data from three to four independent experiments were quantified (lower) to show the density of each GRK remaining after siRNA transfection. (B) Whole cell phosphorylation assays in cells expressing FFA4-L show that agonism with DHA (100 μM, 5 minutes) induces robust phosphorylation of FFA4-L (305.9% ± 23.7% of scrambled control), which is not affected by siRNA knockdown of GRK2 (317.6% ± 7.9%), GRK3 (300.1% ± 19.1%), or GRK5 (297.7% ± 22.8%), but is significantly decreased upon GRK6 knockdown (134.4% ± 28.4%). A representative autoradiograph (upper) is shown while the graph depicts pooled data from three independent experiments (lower). ** P < 0.01 vs. scrambled, DHA-stimulated control (Scr +).