Silencing of CCR2 protein by pSUPER-H1.shCCR2. (A) FACS analysis of HEK 293 cells after cotransfection with pEGFPN-1/CCR2 and pSUPER-H1.Empty (1.5 μg, control), which was not significantly affected by cotransfection with pSUPER-H1.shLuc or pSUPER-H1.shInert. Cotransfection with increasing amounts of pSUPER-H1.shCCR2 led to a significant and dose-dependent silencing of CCR2 protein level up to 75%. *P ≤ .05. (B) The average fluorescence intensity of the GFP-positive cells was even more markedly reduced after cotransfection with pSUPER-H1.shCCR2 to the untransfected HEK 293 control level (approximately 390). The nonspecific controls had no effect on GFP fluorescence intensity compared with pSUPER-H1.Empty control levels. **P ≤ .01; ***P ≤ .001. (C) Western blot analysis of HEK 293–cell lysates for GFP protein expression using β-tubulin (50 kDa) as internal standard, showing a dose-dependent and specific reduction in GFP protein expression in cells cotransfected with increasing amounts of pSUPER-H1.shCCR2 (lanes D, E, and F) compared with only pEGFPN-1/CCR2-transfected (lane B) or pSUPER-H1.Empty cotransfected cells (lane C). Lane 1 represents a sample of untransfected HEK 293 cells. (D) Measurements of JE-induced calcium influx in HEK 293 cells expressing murine CCR2 and the H1.Empty (▴) or the H1.shCCR2 sequence (□). As a control, unstimulated HEK 293 cells expressing CCR2 were measured (•). In contrast to pSUPER-H1.Empty–transfected cells, no calcium influx was observed in pSUPER-H1.shCCR2–transfected cells after stimulation with JE, indicating that CCR2 signal transduction is silenced by shCCR2 treatment. Error bars represent SEM.