Fig. 4
Pim1 regulates c-Kit gene translation. a Pim1 enhances the synthesis of new c-Kit protein. HEK 293 cells transfected with Fu-CRW-Ctl or Fu-CRW-Pim1 were treated with cycloheximide (CHX, 100 µg/ml) for 3 h. The cells were then labeled with 35S methionine. Newly synthesized c-Kit was immunoprecipitated and separated by SDS-PAGE, and visualized by autoradiography (top panel). Aliquots of the cell lysates were run on SDS-PAGE and stained with Coomassie blue to be used for controlling the amount to protein loading (bottom panel). M protein molecular weight marker. Data represent 2 independent experiments. b Pim1 kinase enhances c-Kit RNA incorporation in ribosome fractions. HEK 293 cells were transduced with control lentiviruses (Fu-Ctl) or Pim1-expressing lentiviruses (Fu-Pim1). Light and heavy ribosome fractions were collected for 17 fractions (0.5 ml/fraction) by sucrose gradient centrifugation. The Levels of c-Kit mRNA and β-actin mRNA in the fractions were measured by semi-quantitative RT-PCR assays and the products were run on agarose gel. c Pim inhibitors down-regulated the phosphorylation of eIF4B Ser422. Molm-16 cells were treated with DMSO or pan-Pim inhibitors (SGI1776 at 7 μM or CX6258 4 μM) for 24 h. The cells were harvested and immunoblot analyses were performed. Data represent 3 independent experiments