Figure 8.

PTHrP enhances Src phosphorylation in DRG neurons and increases the TRPV1 protein level in plasma membrane. A, Representative immunoblot, from four independent experiments, showing PTHrP exposure (20 nM, 10 min) leads to Src activation in cultured mouse DRG neurons, as quantified by enhanced phospho-Src (pSrc) levels normalized to total-Src (tSrc) levels shown in panel B. This effect was reduced by co-application of Src inhibitor PP2 with PTHrP. C, Representative immunoblot showing PTHrP exposure (20 nM, 10 min) leads to an increase in TRPV1 protein levels in the plasma membrane (PM) vs intracellular organelle membrane (OM) in lysates of HEK293T cells co-transfected with rTRPV1-WT-myc and rPTH1R-YFP. This effect was absent in the lysates of HEK293T cells co-transfected with the Src phosphorylation site mutant rTRPV1-Y199F-myc and rPTH1R-YFP. Results from multiple experiments were quantified and presented as fraction of plasma membrane TRPV1 protein levels out of normalized total membrane TRPV1 (D). Numbers on the left in panels A and C indicate relative mobility of protein molecular weight markers (in kilodalton; kD). Data are presented as mean ± SEM (n = 4 for panel B, and n = 3–6 transfection batches for panel D). *p<0.05 vs untreated control (One way ANOVA with Dunnett’s post hoc correction, and unpaired Student’s T-test).