Research Article

PKCδ Regulates Translation Initiation through PKR and eIF2α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

Figure 3

PKR regulates phosphorylation (Ser51) of eIF2α. (a) NB4 cells were treated with either ATRA (1 μM) or ATO (0.4 μM) at the indicated time points and PKR levels were detected by Western blot using PKR specific antibody. Densitometry analysis (lower panel) represents relative PKR expression after normalizing to actin expression. (b) NB4 cells were treated with either ATRA (1 μM) at the indicated time points and p-(Thr446) PKR levels were detected by Western blot. (c) HL60 cells were treated with either ATO (0.4 μM) or ATRA (1 μM) at the indicated time points and PERK, p-(Thr981)PERK, p-eIF2α, and PKR levels were detected by Western blot. (d) Expression of GCN2, an eIF2α kinases, is not induced by ATRA in NB4 cells. GCN2 positive cell lysate were used as positive controls for anti-GCN2 antibody and Western blot analysis. (e) ATO and ATRA do not induce PERK eIF2α kinase. NB4 cells were treated with either ATO (0.4 μM) or ATRA (1 μM) at the indicated time points and PERK, p-(Thr981)PERK, p-eIF2α, and PKR levels were detected by Western blot. (f) Knockdown of PKR by a specific siRNA blocks the ATRA-induced phosphorylation of eIF2α in NB4 cells by Western blot. Bar graph represents inhibition and the relative expression of PKR (PKR/β-actin) and p-eIF2α (p-eIF2α/β-actin) after siRNA treatments by densitometry analysis of Western blot bands.
482905.fig.003a
(a)
482905.fig.003b
(b)
482905.fig.003c
(c)
482905.fig.003d
(d)
482905.fig.003e
(e)
482905.fig.003f
(f)