Fig. 8. Depletion of RAMP1 in HEK293 cells alters CaSR traffic. (A) Silencing of RAMP1 using siRNA. HEK293 cells were transfected with 20 nM siRNAs (control, siRAMP1# 1, siRAMP1 #2, see Materials and Methods) and 500 ng Myc-RAMP1. Whole cell lysates were prepared 48 hours after transfection. The immunoblot was probed with anti-Myc antibody and reprobed with anti-ß-actin antibody to ensure equal loading. Films were scanned and quantified using the image J software. A significant decrease of RAMP1 expression (*P<0.05) was observed with siRAMP1 #2 compared to the expression level in the control. The blots shown are representative of three independent experiments. (B) Silencing RAMP1 alters CaSR traffic. HEK293 cells were transfected with 20 nM Cy3-labelled siRNA against RAMP1 (siRAMP1 #1 and #2) or control siRNAs together with 200 ng pf pRK5-SEP-CaSR. 48 hours after transfection, the surface expressed SEP-CaSR population was labelled with Alexa 655 anti-GFP antibody, cells were fixed and imaging was performed as described in Materials and Methods. SEP-CaSR appears green, Cy3-siRNAs red and surface CaSR blue. Bar, 5 µm.