Sertoli cells were cultured for 4 days prior to their use for this experiment as described in Materials and Methods. Culture medium (F12/DMEM) was supplemented with TNFα (20 ng/mL) on day 5 and thereafter. Cultures were terminated at specified time points and processed for immunoblot analysis. A: CAR protein level decreased by 2 to 3 days after TNFα treatment, whereas the level of occludin and JAM-A dropped significantly by 4 hours after TNFα treatment. B: Bar chart summarizes results from 3 sets of immunoblot using different batches of Sertoli cell cultures. The steady-state protein level of the target protein at time 0 was arbitrarily set at 1, against which one- way ANOVA was performed. ns, not significantly different *, P<0.05 **, P<0.01