Horseradish peroxidase conjugated anti mouse antibody

5-Hydroxytryptamine hydrochloride (5-HT, serotonin), nystatin, dynasore hydrate, brefeldin A, filipin, TX-114, digitonin anti-HA/myc antibodies (Sigma), 2-methyl-5-Hydroxytryptamine hydrochloride (Tocris, Bristol, UK), [³H]BRL-43964 ([³H]granisetron), and 5-[³H]HT (PerkinElmer Life Sciences). Cell culture reagents, Amplex UltraRed, and Alexa Fluor 488/568 conjugated anti-mouse antibody (Invitrogen). Horseradish peroxidase conjugated anti-mouse antibody (GE Healthcare).

For assessment of surface and total expression of WT and mutant CaSR constructs, ELISA was performed as previously described44 (link). In brief, cells were fixated using 4% paraformaldehyde in DPBS for 5 min at RT. For measurements of total expression, cells were permeabilized using 0.1% Triton-X in DPBS. All wells were incubated in blocking solution (ddH₂O with 3% skim milk, 1 mM Ca²⁺, 50 mM Trizma hydrochloride solution, pH 7.4) for 30 min after which primary antibody incubation for 45 min at RT was performed by addition of either anti-HA antibody (Nordic BioSite, Täby, Sweden) or anti-myc antibody (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:1000 in blocking solution. Subsequently, horseradish peroxidase conjugated anti-mouse antibody (GE Healthcare Biosciences, Pittsburgh, PA, USA) diluted 1:1500 in blocking solution was added and plates were incubated for 45 min at RT. Receptor levels were detected by addition of 80 μl/well of DPBS supplemented with 1 mM CaCl₂ and 10 μl/well of SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Chemiluminescence was measured on an EnSpire plate reader (PerkinElmer, Waltham, MA, USA) and normalized to the level of WT CaSR.