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. 2015 Aug 31;112(37):E5160–E5168. doi: 10.1073/pnas.1508836112

Fig. 2.

Fig. 2.

Activation of the ERK/MAPK pathway reduces cell-surface expression of CXCR4. (A) Schematic representation of the receptor cell-surface expression assay. PM, plasma membrane. (B and C) Cell-surface CXCR4 levels were detected in HEK293T cells stably expressing HA-CXCR4-vYFP transfected without (Control) or with the indicated Ras and MEK mutants. Cells were labeled with a primary anti-HA and secondary Alexa 647-coupled chicken anti-mouse IgG antibodies, without permeabilization. Cell-surface expression of HA-CXCR4-vYFP in the absence of CXCL12 stimulation was measured by dual-flow cytometry. vYFP emission represents CXCR4 total expression; Alexa Fluor 647 emission represents CXCR4 plasma membrane expression. The relative cell-surface expression (the ratio of Alexa:vYFP mean emissions) is calculated from the YFP+ cell population and is expressed as a percentage of the control condition. Data shown represent the mean ± SEM of three independent experiments. Expression of CA or DN forms of Flag-Ras or Flag-MEK and total ERK and ERK1/2 activation status [(p)ERK1/2] was assessed by immunoblotting. Representative experiments are shown below the graphs. **P < 0.01; NS, not significant.