Figure 2

<p><b>Sequential phosphorylation at CXCR4 C-terminal sites. </b><b>A–F</b>, Stably CXCR4-transfected HEK293 cells were stimulated with 20 nM CXCL12 for 0, 2, 5, and 15 min. Aliquots of the lysates were detected in four immunoblots using the indicated antibodies. Blots were stripped and reprobed with anti-HA and anti-transferrin receptor (TFR) to control for equal loading. <b>A</b>,<b>B</b>, CXCL12 induces a rapid loss of the anti-S341-S352 signal and a concomitant immediate increase of the anti-pS346/347 signal. <b>C</b>, Maximal S338/339 phosphorylation requires 15 min CXCL12 stimulation. <b>D</b>, Demonstration of rapid CXCL12-promoted S324/325 phosphorylation. <b>E</b>–<b>I</b>, The signal ratio versus anti-HA was determined for the four anti-CXCR4 antibodies using densitometry. The ratio was set as 100% at 0 min for anti-S341-S352 (G) and at 15 min for the phospho-selective antibodies (G–I). Data represent mean+SEM from 4–5 independent experiments. <b>G</b>, Note complementarity of the anti-S341-S352 and anti-pS346/347 time courses. <b>H</b>,<b>I</b>, Phosphorylation occurs faster at S346/347 than at S338/339 and S324/325. *: p<0.05; 1way ANOVA and Bonferronís multiple comparison post-test for selected groups.