PMLRARα blocks CH-11 antibody and Fas ligand binding to Fas. (A) U937/PR9 cells were treated with or without ZnSO₄ for 24 hours to induce expression of PMLRARα and subsequently incubated with agonistic anti-Fas antibody CH-11. Unbound antibody was removed by washing. Cells were lysed and precipitated with goat anti–mouse IgM antibody to coprecipitate CH-11–bound Fas (Active Fas, top panel). Total cell extracts were also Western blotted with anti-Fas and anti-RARα antibodies for detection of Fas and PMLRARα. (B) Expression of PMLRARα was induced as in panel A and cells were incubated with FasL. Excess of FasL was washed off and cells lysed. FasL complexes were precipitated to detect activation accessible Fas. (C) Scrambled shRNA expressing cells and NB4 clone 4 cells were incubated with FasL as in panel B. Cell extracts were used to immunoprecipitate FasL to detect FasL-bound/activation accessible Fas (top panel). Whole cellular levels of Fas and PMLRARα were analyzed by Western blot. (D) U937/PR9 cells were treated with 0, 100, and 200μM ZnSO₄ for 24 hours and stained with anti-Fas antibody, to evaluate cell surface Fas levels by flow cytometry as described in “Flow cytometry analysis of surface Fas expression level.”