Fig. 2

Ate1 associates with microsomal membranes under proteasomal inhibition. a Human oligodendroglioma (HOG) cells were treated with 500 nM BT for 16 h. Cell lysates were subjected to ultracentrifugation at 100,000 rpm to separate them into WL, supernatant (S₁₀₀) and precipitate (P₁₀₀) fractions. Cav1 and Gapdh were used as endogenous controls for membrane-bound and soluble proteins, respectively. (right panel) Quantification of Ate1 levels in the P₁₀₀ fraction relative to the S₁₀₀ fraction. Statistical significance was assessed using a two-sided unpaired t test. Quantification represents the mean ± SE of four independent experiments. *P≤0.05. b Properties of Ate1 association with membranes. The S₁₀₀ and P₁₀₀ fractions from BT-treated HOG cells were prepared as described in (a). The P₁₀₀ fraction was then incubated for 40 min with Tris buffer or 0.1 M Na₂CO₃. After treatment, solubilized proteins (sol) were separated from those remaining bound to membranes (ins) by ultracentrifugation at 100,000 rpm and analyzed by Western blotting. Cav1 and Gapdh were used as markers for non-extractable membrane protein and soluble protein, respectively. c Detergent partitioning of Ate1 into the S₁₀₀ and P₁₀₀ fractions. The S₁₀₀ and P₁₀₀ fractions from HOG cells treated with BT, obtained by ultracentrifugation at 100,000 rpm, were incubated with TX114 and partitioned into the aqueous phase (aq) or detergent (det). The fractions were subsequently analyzed by Western blotting. Cav1 and Tubulin were used as endogenous controls for the membrane-bound and soluble proteins, respectively. (a-c) Endogenous Ate1 levels were detected using an antibody against Ate1