Fig. 3
Western blot analysis and negative-stain electron microscopy of purified TRPC3. TRPC3 was purified from adherent HEK293 cell membranes using DIBMA (A), K. phaffi cell membranes using DDM (n = 3, B), and Expi293F cell membranes using DDDG (n = 6, C) or DDM (n = 6, D). The resulting concentrations of purified TRPC3 are shown in (E). TRPC3 protein concentration (Vtotal = 150 µL) was lower from K. phaffi cell membranes compared to adherent HEK293 or Expi293F cells. Specifically, TRPC3 concentration was 0.05 mg/mL from K. phaffii cell membranes using DDM, 0.2 mg/mL from HEK293 cell membranes using DIBMA, and 0.15 mg/mL from Expi293F cells using either DDM or DDDG. Negative-stain electron microscopy images of TRPC3 extracted with DDM (F) or DDDG (G) from Expi293F cells reveal the tetrameric structure of TRPC3 channels (blue arrows). The blue box highlights the magnification of a single TRPC3 tetramer as observed in the electron microscopy images. H) General cryo-EM structure of the closed homomeric TRPC3 channel from the side (left) and top (right) view (PDB: 7DXB)42. Note: Please note that the Western blots in Panel A-D were cropped. For the full, uncropped Western blot, refer to Supplementary Information, Figure S7.
