VR1 dimerization is not affected by reducing agents, capsaicin, Ca2+ or transglutaminase inhibitors. (A) Western Blot of the effect of reducing agents on the VR1 dimer. The addition of DTT (100 mM) and TCEP (20 mM) did not modify the ratio of monomer to dimer in VR1-expressing oocytes (p > 0.05, for 3 independent experiments). (B) Effect of Ca2+ on dimer formation in VR1. The addition of Ca2+ to the biochemical assays (in the presence or the absence of capsaicin, lanes 1 and 2) did not modify the ratio of monomer to dimer (p > 0.05, for 3 independent experiment). Addition of EGTA (2 mM) to the assays (lanes 3 and 4) did not modify the monomer to dimer ratio either (p > 0.05, for 3 independent experiments). For the Ca2+-free condition, the expected free Ca2+ concentration was 0.4 nM, calculated using WebMax C version 2.1 http://www.stanford.edu/~cpatton/webmaxc2.htm. and assuming a contaminant level of 5 μM Ca2+ in our water. For the condition in which Ca2+ was present, we added 1.8 mM Ca2+ and no chelator to the solution (see Materials and Methods). (C) Effects of transglutaminase inhibitors on dimerization of VR1. The addition of cysteamine (20 mM) and MDC (250 μM) to oocytes did not alter the amount of dimer in relation to monomer when compared to the control lane which did not receive any treatment (p > 0.05, for 3 independent experiments).