Protease protection assay. FKBP65 is entirely
intravesicular. The 200,000g microsomal pellet from
NIH3T3 cells was treated with buffer alone (control), 100 μg/ml
proteinase K alone, or 100 μg/ml proteinase K + 1% Triton X-100.
After 30 min at room temperature, excess PMSF was added to each sample
to inhibit the enzyme, and the membranes in all samples were
solubilized with Triton X-100. Proteins were acetone precipitated and
Western blotted. CNX-CT = calnexin C-terminal antibody.