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. 2014 Jan 8;289(9):5820–5827. doi: 10.1074/jbc.M113.508440

FIGURE 3.

FIGURE 3.

The CSF3R T618I mutation caused increased receptor dimerization. A, schematic of CSF3R dimerization studies. The CSF3R wild type and T618I constructs (0.15 μg) with either FLAG (F) or V5 (V) tags were cotransfected into 293T17 cells, and an anti-FLAG antibody was used to immunoprecipitate FLAG-tagged CSF3R. Dimerization was then assessed by immunoblot analysis with a V5-specific antibody B, FLAG- and V5-tagged CSF3R wild type or T618I constructs were cotransfected into 293T17 cells. The amounts of receptor expression from each pair of constructs were comparable between the wild type and T618I as assessed by immunoblot analysis. For each pair of constructs, 0.3, 0.15, or 0.075 μg of plasmid was transfected. Anti-FLAG affinity resin was used to immunoprecipitate (IP) FLAG-tagged CSF3R from whole cell lysates. Immunoblot analysis using an anti-FLAG antibody was used to detect the levels of FLAG pulldown. Immunoblot analysis using an anti-V5 antibody was used to detect coimmunoprecipitating CSF3R, indicative of CSF3R dimerization. C, quantification of the immunoblot analysis in B. The levels of direct FLAG immunoprecipitation and V5 coimmunoprecipitation were quantified using ImageJ for each plasmid concentration. The Ratio of V5 to FLAG is shown normalized to the respective input levels. Increased dimerization of the CSF3R T618I construct relative to the CSF3R wild type was seen at each plasmid concentration. D, FLAG- and V5-tagged CSF3R wild type or T618I constructs were cotransfected into 293T17 cells as either WT/WT, T618I/T618I, WT/T618I, or T618I/WT pairs. The ratio of V5 to FLAG is shown normalized to the respective input levels. Three replicates were performed for each condition, and the mean ± S.E. are shown. Increased dimerization of the CSF3R T618I construct relative to the CSF3R wild type was found to be statistically significant, although there was a trend of increased dimerization of WT/T618I and T618I/WT over WT/WT, but this was not statistically significant. Values represent mean ± S.E., n = 3. Statistical significance was assessed using one-way analysis of variance followed by Bonferroni's multiple comparison test. **, p < 0.01. This experiment was performed three times with consistent results.