Anti calnexin

Western blot analysis was performed as previously described (Gorvin et al. 2018d (link)). Endogenous calnexin was used as a loading control. Lysates were resuspended in Laemmli buffer, boiled and separated on 6% SDS-PAGE gels. Following transfer to polyvinylidene difluoride membrane (ThermoFisher), blots were blocked in 5% marvel/TBS-T, then probed with anti-CaSR (ADD, Abcam), anti-Gα11 (SantaCruz Biotechnology), anti-Gα12 (SantaCruz), and anti-calnexin (Millipore) antibodies. Blots were visualised using the Immuno-Star WesternC kit (BioRadK) on a BioRad Chemidoc XRS+ system. For cell surface expression of CaSR, plasma membrane fractions were extracted using the Plasma Membrane Protein Extraction kit (Abcam). Plasma membrane calcium adenosine triphosphatase (PMCA1) (Abcam) was used as a housekeeping protein for the plasma membrane fraction.

Following flow cytometry analysis, cells were pelleted and used for Western blot analyses. Flow cytometry cells and lymphoblastoid cells were lysed in NP40 lysis buffer (50 mM Tris HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, protease inhibitors), resuspended in Laemmli buffer, boiled, and separated on 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels. Following transfer to polyvinylidene difluoride membrane (Amersham), blots were blocked in 5% nonfat milk/TBS‐T, then probed with primary antibodies. The following antibodies were used for Western blot analysis: anti‐calnexin (1:1000, Millipore, AB2301), anti‐Gα₁₁ (1:1000, SantaCruz, sc‐390382), and anti‐GFP (1:1000, SantaCruz, sc‐9996). Blots were visualized using Immuno‐Star WesternC kit (BioRad) on a BioRad Chemidoc XRS+ system. Blots were stripped with Restore Western blot stripping buffer (Thermo Fisher Scientific) and blocked in 5% marvel/TBS‐T between probing with each primary antibody. Densitometric analysis was performed using ImageJ analysis software (version 1.46; rsb.info.nih.gov/ij/), and statistical analyses were performed using one‐way ANOVA with Dunnett's multiple comparisons test.⁽⁶ (link)
⁾ Expression levels of Gα11 were normalized to calnexin expression and expressed relative to the mean relative density of the three unaffected cell lines.

Cells were transfected with 2 µg of each construct. Control
experiments were performed by transfecting the empty vectors. Forty-eight hours
after transfection, cells were subjected to cell-surface biotinylation and
precipitated after lysis with neutravidin–agarose beads (Pierce Rockford, IL,
USA) as described in.^{21 (link)} Anti-TRPV1 antibody (1/500, Santa Cruz)
and anti-calnexin (1/2000, Millipore) were used.