ERK knockdown does not reduce C/EBPα expression or impair granulopoiesis. (A) 32Dcl3-Puro (32D), 32Dcl3(shRNA-77)-10, or 32Dcl3(shRNA-77)-12 cells were removed from IL-3 for 3 hours and then exposed to G-CSF for 0, 5, or 10 minutes. Total cellular proteins were then subjected to Western blotting for phosphorylated ERK (P-ERK) or for total ERK levels. (B) Total cellular proteins from 32Dcl3 cells transduced with the pLKO.1-Puro and pLKO.1-Neo vectors (P/N) or from 2 subclones of 32Dcl3 cells (ERKshR-a and ERKshR-b) expressing shRNAs targeting ERK-1 and ERK-2 were subjected to Western blotting for ERK, P-ERK, C/EBPα, and β-actin. (C) Growth curves show daily viable cell counts for the cells transduced with empty vectors and for the 2 subclones with knockdown of ERK-1 and ERK-2. (D) Total cellular RNAs prepared from these 3 cell lines 1 day after transfer to G-CSF were analyzed for relative expression of MPO and PR3 RNAs. The average expression of PR3 RNA was set to 1.0 in the 32Dcl3-Puro/Neo cells. Results shown are mean and SE from 3 determinations. (E) 32Dcl3-ERK shRNA subclones ERKshR-a and ERKshR-b were cultured in G-CSF for 4 days, cytospun, and subjected to Wright-Giemsa staining.