Fig. 9.

Plasma membrane expression and oligomeric assembly of rASIC3 and rP2X3R subunits in oocytes. For biochemical analysis, Xenopus (X.) laevis oocytes expressing the indicated proteins were surface labeled with the membrane-impermeant fluorescent Cy5 dye before protein purification. The indicated proteins were purified under non-denaturing conditions from X. laevis oocytes by Ni²⁺-NTA chromatography or Strep-Tactin chromatography, as indicated, resolved by SDS-PAGE (a) or BN-PAGE (b), and visualized by Typhoon fluorescence scanning. a Overlay of the Cy5-labeled surface form (red) of the His-rASIC3-EGFP and His-rP2X3-StrepII protomers and the GFP fluorescence (green) of the His-rASIC3-EGFP protomer. The positions of molecular mass markers (in kDa) are shown on the left. b Overlay of GFP and Cy5 fluorescence of the homomeric His-rASIC3-EGFP (lanes 1–4) and homotrimeric His-rP2X3-StrepII (lanes 15–18) and the co-expressed His-rASIC3-EGFP and His-rP2X3-StrepII receptor subunits (lanes 5–14) in their native or partial SDS-denatured forms isolated by Ni²⁺-NTA (lanes 3–6, 10, 11, 15, 16) or Strep-Tactin (lanes 7–9, 12–14, 17, 18) chromatography as indicated. Co-expression of rASIC3 and rP2X3 (lanes 5–14) originates from the co-injection of the His-rASIC3-EGFP and His-rP2X3-StrepII subunit in cRNA ratios as indicated. The number of protomers included in the respective bands are exemplarily indicated. The inset shows the indicated section of the gel with enhanced GFP fluorescence to enable the visibility of the ASIC3 trimer. The schematics and labeling on the left or right margins indicate the numbers of rASIC3 or rP2X3 protomers incorporated in the respective trimeric (bold) or dimeric or monomeric protein band of the rASIC3 or rP2X3 receptor complexes, respectively