HPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha
Figure 2
Time course of changes in HPV16 E7 expression and both phosphorylation and activity of p38 MAPK in 2BN11 cells after tetracycline (tet) removal.
A. The activation of HPV16 E7 transcription upon tet removal from the culture medium was determined by RT-PCR as described in in Materials and Methods. After tet removal the cells were collected at the indicated times, total RNA prepared, cDNA generated and the levels of message were determined. The levels of GAPDH were also determined in each sample as internal control. nc: negative control in which the PCR was performed in the absence of a template; pc: positive control included HPV16 E7 cDNA as template. B. Time course of HPV16 E7 expression-dependent inhibition of p38 phosphorylation, total p38 and tubulin in 2BN11 cells after tetracycline removal. C. p38 activity during HPV16 E7 expression (tet removal) was determined by exposing p38 immunoprecipitated from 1 mg of cell lysate to 10 µg GST-ATF-2 as described in methods. The amount of ATF-2 phosphorylated by p38 was analyzed by Western Blotting with an Phospho-ATF-2 (Thr71) antibody and tubulin levels in the homogenate was used as loading control.