Fig. 6. Direct interaction between endogenous Cx43 with Cx43/ß-gal. (A) Membrane preparations from control (lanes 1, 3), 43ß1 (lanes 2, 4) and 43ß2 cells (lane 5) were solubilized in 1% Triton X-100 at 4°C for 30 minutes and then centrifuged at 100,000 g for 30 minutes to remove insoluble material. The supernatant was then incubated with mouse anti-ß-gal antiserum and goat anti-mouse IgG-conjugated magnetic beads, then magnetically isolated and solubilized in SDS-PAGE sample buffer. The resulting samples were resolved by electrophoresis, immunoblotted using rabbit antisera which recognizes either ß-gal (lanes 1, 2) or Cx43 (lanes 3-5), then detected by chemiluminescence. The arrowhead indicates bands corresponding to Cx43/ß-gal, while the dot indicates endogenous Cx43. (B,C) Membrane-enriched fractions from control cells (B) or 43ß2 cells (C) were solubilized in Triton X-100 and then analyzed by sucrose gradient fractionation as described in Materials and Methods. The solid line indicates fractions containing unmodified, endogenous Cx43 while the broken line in C corresponds to Cx43/ß-gal. Region 1 of the gradient (5%-11% sucrose) corresponds to Cx43 monomers, while Cx43 hexamers sediment at region 3 of the gradient (approx. 15%-18% sucrose), peaks that correspond to 5S (HRP) and 9S (catalase) standards, respectively. Expression of Cx43/ß-gal caused an increase in Cx43 which cosedimented in region 2 of the gradient (11%-15% sucrose). Region 4 of the gradient (18%-20% sucrose) from 43ß2 cells contained some Cx43/ß-gal complexes, but showed little, if any native Cx43.