Figure 8.

Translocation of sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA2) within caveolae-related domains (CRDs) from the estimation of endoplasmic reticulum (ER) of cystic fibrosis (CF) cells. Purified microsomes from 16HBEo- and CF45o- cells were lysed in ice-cold 0.5 M sodium carbonate buffer. The homogenate was adjusted to 45% (w/v) sucrose by the addition of 90% sucrose in the MBS buffer and placed in the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient was established by overlaying this solution with 4 ml of 38% sucrose and 3 ml of 5% sucrose. The tubes were then centrifuged at 4°C for 16 to 18 hours at 130,000 × g, and fractions were manually collected from the top of the gradient. To determine the distribution of CRD-associated proteins within the gradient, each fraction was analyzed by SDS-PAGE, followed by Western blot analysis with SERCA2 and caveolin antibody (A). The Western blot shown is representative of findings in three separate sucrose gradient centrifugation/Western blot experiments. (B and C) Western blot of SERCA2 and Bcl-2 using immunoprecipitate of microsomal fractions from (1) 16HBEo- and (2) CF45o- using Bcl-2 antibody.