Fig. 5. Time course studies on the effect of TNF+CHX treatment on caspase 3 activation and MHCIIA degradation in wild type and MHCIIB^–/– embryonic fibroblasts. Wild type and MHCIIB^–/– cells were incubated in the absence (0 hour) or in the presence of TNF+CHX for increasing periods of time (2-24 hours). (A) Total cell lysates were subjected to western blotting analyses: the same membrane was incubated with antibodies against active caspase 3, MHCIIA and actin. The figure shows representative western blots. The time course of caspase 3 cleavage and of MHCIIA disappearance was similar in the two cell lines. (B) Following treatments, cells were processed for immunofluorescence microscopy with anti-active caspase 3 and Rhodamine-phalloidin. Total adherent cells and adherent cells positive for active caspase 3 were counted for each time point. TNF+CHX treatment induced an increase in the percentage of cells positive for active caspase 3 with the same time course in both cell lines. The values shown are the mean ± s.e.m. of three independent experiments and more than 300 cells were observed per experimental condition.