Figure 1.

FLAG-MOP/TRPV1 double stable HEK293 cells express TRPV1 and MOP. (A) Western Blot Analysis of TRPV1 expression from HEK293 cells stably expressing TRPV1 and FLAG-tagged MOP. Precipitated cell proteins were subjected to SDS PAGE on a 7.5% gel, transferred to nitrocellulose membrane and probed for TRPV1 expression using a N-terminal directed TRPV1 antibody. β-actin was used as a loading control. Expected bands for the TRPV1 are at approximately 95 kDa for the unglycosylated and 113 kDa for the glycosylated receptor. Lane 1: Colony 13 FLAG-MOP/TRPV1 double expressant; Lane 2: Colony 21 FLAG-MOP/TRPV1 double expressant; Lane 3: untransfected HEK293 cells; Lane 4: FLAG-MOP only transfected HEK293 cells. (B) Western Blot showing FLAG-MOP protein in the same FLAG-MOP/TRPV1 and FLAG-MOP only cells, determined using an anti-FLAG M2 antibody. Expected protein size was a broad band at approximately 60 kDa representing multiple glyocsylated FLAG-MOP species. Lane 1: Colony 13 FLAG-MOP/TRPV1 double expressant; Lane 2: Colony 21 FLAG-MOP/TRPV1 double expressant; Lane 3: FLAG-MOP only transfected HEK293 cells; Lane 4: untransfected HEK293 cells. Representative blots from at least 3 independent experiments are presented. (C–E) FLAG-MOP/TRPV1 cells co-express TRPV1 and FLAG-MOP. Double stable FLAG-MOP/TRPV1 cells were stained for FLAG-MOP (D, green) and TRPV1 (E, red) expression with the overlay (C, yellow) clearly demonstrating co-expression of the two receptors. Specificity of the antibodies used was demonstrated by lack of non-specific binding (F, red and G, green). Scale bar, 40 μm.