Heterologous and homologous phosphorylation of CXCR4.

<p><b>A</b>, Stably CXCR4-transfected HEK293 cells were stimulated for 30 min as indicated to produce maximal effects. Note that CXCL12 and PMA but not EGF or forskolin induce CXCR4 phosphorylation. <b>B</b>, When indicated, cells were pre-incubated for 30 min with the non-selective PKC inhibitor staurosporine and stimulated for 15 min with CXCL12 or PMA in the presence or absence of staurosporine. Staurosporine reduces CXCL12-promoted phosphorylation at S324/325 and reduces PMA-induced phosphorylation at all three sites. <b>A</b>,<b>B</b>, Images are representative for three independent experiments yielding similar results. <b>C</b>,<b>D</b>, Cells were pre-treated for 72 h with control siRNA (scr) or siRNAs for GRK2 and GRK3 as indicated. Lysates were generated 15 min after CXCL12 stimulation and detected in an immunoblot with anti-pS346/347 and anti-HA. Combined knockdown of GRK2 and GRK3 reduces the CXCL12-promoted signal increase of anti-pS346/347 indicating attenuated phosphorylation at S346/347. <b>D</b>, Quantification of 3 independent experiments analyzing the effect of GRK2 and GRK3 knockdown on CXCL12-promoted S346/347 phosphorylation. *: p<0.05; Student's t test vs. scr-group. <b>E</b>, Western Blot analysis of the siRNA-mediated knockdown of GRK2 and GRK3.