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Figure 4


Fig. 4. Effect of TNF{alpha}+CHX treatment on MHCIIA and MHCIIB levels in wild-type and MHCIIB–/– embryonic fibroblasts. (A) Wild-type and MHCIIB–/– embryonic fibroblasts were treated with different combinations of TNF{alpha} (20 ng/ml), CHX (5 µg/ml), Z-VAD-fmk (5 µM) and Y-27632 (20 µM) for 24 hours. Following treatments, total cell lysates were subjected to western blotting with antibodies against MHCIIA, MHCIIB, spectrin, actin and active caspase 3. TNF{alpha}+CHX treatment reduced MHCIIA levels and caused the cleavage of spectrin and caspase 3 in both wild-type and MHCIIB–/– cells. Control conditions for each protein were restored by the caspase inhibitor Z-VAD-fmk but not by Y-27632. MHCIIB expression in wild-type cells was not affected by the treatments. The figure shows representative western blots. (B) Quantification of MHCIIA levels in wild type and MHCIIB–/– fibroblasts. The intensity of the MHCIIA immunoreactive bands was measured for each experimental condition described in A and normalised to control cell levels. Values shown are the mean ± s.e.m. of three independent experiments. The graph shows that TNF{alpha}+CHX treatment had a similar effect on MHCIIA levels in both cell lines. *P<0.01: wild-type fibroblasts, TNF{alpha} vs TNF{alpha}+CHX; **P<0.0002: MHCIIB–/– fibroblasts, TNF{alpha} vs TNF{alpha}+CHX.





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