Fig. 2. Pim inhibition downregulates Mcl-1 expression by blocking protein synthesis.

A, LNCaP cells expressing shRNA constructs targeting Pim-1, 2 and 3, nonsilencing control (shC) or treated with SMI-4a (10 µM) for 16h were lysed and cell extracts were analyzed by immunoblotting with the antibodies listed. B, LNCaP cells were treated with varying doses of SMI-4a for 16h or with SMI-4a (10 µM) for different times. Cell extracts were analyzed by immunoblotting with the antibodies shown. C, LNCaP cells were methionine starved, treated with DMSO or SMI-4a for 16h, and incubated with ³⁵S-methionine. After labeling, the Mcl-1 protein was immunoprecipitated, and analyzed by autoradiography and immunoblotting. The arrow denotes the upper band that is identified as the Mcl-1 protein. D, Aliquots of whole cell lysates obtained in C were washed with 10% TCA, the protein dried, and the proteins labeled with ³⁵S-methionine quantified by scintillation counting (mean +/− SD, n=6). The p value of p < 0.05 for differences between DMSO and SMI-4a treatment was obtained by a two-way analysis of variance.