Figure 3

Temperature shifting does not affect the cell cycle or TNF-α signaling. (A) 4Bv cells were maintained at 37°C or 32°C overnight, followed by treatment with or without TNF-α for 6 h. Flow-cytometric analysis was performed on DAPI/SR 101 stained cells to determine the percentage of 4Bv cells in different cell cycle phases (G1, S, G2/M). Mean values (columns) and standard deviations (bars) are given for three independent experiments. Steady state expression (B) and phosphorylation (C) of p38 MAP kinase was analyzed after treatment of 4Bv cells (at 37°C or 32°C) with TNF-α as indicated. Total cellular protein (50 μg) was separated in a 12% SDS-PAGE gel. After electrotransfer, the filter was consecutively incubated with antibodies as indicated and re-probed with anti-actin antibody as loading-controls. (-): untreated control cells; (15')/(30')/(60'): cells treated with 250 U/ml of TNF-α for the indicated time.