FIG. 4.

PKCζ is necessary for ceramide-induced inhibition of Akt phosphorylation in L. donovani-infected cells. (A) PKCζ inhibits the phosphorylation of Akt. Macrophages were pretreated with or without cell-permeative PKCζ inhibitor peptide (100 nM) for 1 h and then challenged with L. donovani promastigotes (LD) (cell/parasite ratio, 1:10) for 4 h, noningested promastigotes were removed, and macrophages were cultured for another 20 h. LPS (1 μg/ml) was added where indicated at 30 min prior to the lysis of cells. The lysates were immunoprecipitated with anti-Akt antibody and subjected to immunoblotting with p-Akt Ser473 and p-Akt Thr308 antibodies. The blots were reprobed with anti-Akt antibody to confirm equal protein loading. The lane numbers above Western blots correspond to the x axis of the densitometric analysis below. The data shown in panel B are the means ± SD from four separate experiments. The arbitrary values shown were obtained by densitometric analyses of the protein bands. Asterisks indicate statistically significant inhibition (*, P < 0.05) compared to normal macrophages, restoration (**, P < 0.003) compared to infected macrophages, and inhibition (***, P < 0.001) of phosphorylation of Akt compared to LPS-stimulated macrophages.