Fig. 2.

Mechanisms of MafK protein regulation by NGF. A, Time dependence of effect of NGF on MafK protein levels in PC12 cells. Total cellular protein isolated from PC12 cells was analyzed by Western immunoblotting with ERK and MafK antisera. Comparable results were achieved in two or three independent experiments. B, Effect of the transcriptional inhibitor actinomycin D on regulation of MafK protein by NGF. Cells were pretreated for 90 min with 10 μm actinomycin D (AD) and then for 2 hr with NGF. Total cell protein was subjected to Western immunoblotting with ERK and MafK antisera. Band intensities were determined by densitometry (using shorter times of exposure than shown and for which bands were not at a level of saturation), and values were normalized to ERK signals. Values are mean ± SEM (n = 9). C, Effect of NGF and TPA on MafK protein levels. PC12 cells were treated with no additive (c) or NGF, 50 nm TPA, or both for 2 hr, and then Western immunoblotting was performed with ERK and MafK antisera. Comparable results were achieved in two or three independent experiments. D–F, Effect of signal transduction inhibitors and activators on NGF-promoted MafK levels. PC12 cells were treated for 2 hr with no additive (c), EGF (E), 50 nm TPA (T), or NGF (N) as indicated. Inhibitors (MEK inhibitor U1026, PI3K inhibitor LY294002, and PLCγ inhibitor U73122) were added 1 hr before NGF where indicated. Total cellular protein was isolated and subjected to Western immunoblotting with ERK and MafK antisera. Comparable results were achieved in two or three independent experiments.