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Fig. 6. BPGAP1 and EEN stimulate EGF receptor endocytosis and ERK1/2 phosphorylation. (A) 293T cells were transfected with vector control or expression plasmids coding for the tagged wildtype or mutant proteins, either alone or in combinations as indicated. They were made quiescent by serum depletion for 2 hours followed by stimulation with 100 ng/ml Oregon-Green-labeled EGF for the times indicated and processed for EGF uptake in the presence of monensin as described in the Materials and Methods. The total amounts of EGF-bound EGF receptor being internalized within certain populations of cells that had expressed specific tagged proteins were measured by fluorescence-based detection and calculated as mean intensity per cell, as described in Materials and Methods. Data are means of two to three independent experiments, each counted in triplicate. Variations for each specific time point and sample were typically less than 10% and the error bars are omitted for clarity. The control uptake exhibits a linear uptake rate up to 30 minutes. (B) 293T cells were transfected with vector control or expression plasmids coding for the tagged wildtype or mutant proteins, either alone or in combination as indicated. They were made quiescent by serum depletion for 18 hours followed by the stimulation with 100 ng/ml EGF for the periods indicated. Activation of the EGF receptor and ERK1/2 were analyzed by western blotting of equal amount of whole-cell lysates using anti-phospho EGF receptor and anti-phospho ERK1/2, respectively. To show equal loading of whole-cell lysates and expression of the appropriate tagged proteins, the blots were analyzed with anti-EGF receptor (EGFR), anti-ERK1/2 or anti-Flag (for EEN and NT in single or double transfectants, and for BPGAP1, PP and R232A in single transfectants), or anti-HA (for BPGAP1, PP and R232A in double-transfectants). For triple transfectants, anti-Myc (for EEN), anti-HA (for PP) and anti-Flag (for BPGAP1) were used. The ERK phosphorylation signals were expressed as number of fold over the maximal level seen by the control cells after 2 minutes. These results are representative of three independent sets of assays, each time with the control-stimulated cell lysates prepared concurrently and analyzed alongside the test constructs. This serves as an internal control for gel variations and also for normalizing the film exposures. One of the representative set is shown as Fig. 6C.
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