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. 2015 Jan 20;290(11):6810–6824. doi: 10.1074/jbc.M114.606699

FIGURE 5.

FIGURE 5.

Depletion of ESCRT-I subunit Tsg101 attenuates CXCR4-promoted phosphorylation of Akt at Ser-473. A, BAEC treated with siRNA directed against Tsg101 or GAPDH (Ctrl) were serum-starved in DMEM containing 20 mm HEPES for 3 h, followed by treatment with vehicle (minus symbol) or CXCL12 (plus symbol) for 10 min. Equal amounts of whole cell lysates were immunoblotted for pAkt-S473 and the indicated proteins. Representative immunoblots from three independent experiments are shown. The asterisk likely represents a nonspecific band. B, densitometric analysis of pAkt-S473 levels. pAkt-S473 levels were normalized to total Akt levels, and bars represent the average levels of pAkt-S473 compared with control (Ctrl) and CXCL12-treated cells (set to 100%) from three independent experiments. The error bars represent mean ± S.E. Data were analyzed by a two-way ANOVA followed by Bonferroni's post hoc test (p < 0.05). C, time course of Akt activation promoted by CXCR4 in BAEC cells. Cells were treated with vehicle (time 0) or CXCL12 for the indicated times, and equal amounts of whole cell lysates were immunoblotted for pAkt-S473 and total Akt. Immunoblots were analyzed by densitometry, and the graph shows the average pAkt-S473 levels normalized to total Akt at each time point compared with control (10-min time point; set to 100%) from three independent experiments. Error bars represent S.E.