Forced expression of PDI suppresses CEBP proteins. (A-C) Western blot analyses of subcellular fractions from lung cancer cells H1299 cells transiently transfected with pcDNA3/PDI-V5-His and pcDNA3/CEBPA (A), pcDNA3/PDI-ΔKDEL-V5-His and pcDNA3/CEBPA (B), and pcDNA3/PDI-V5-His or pcDNA3/PDI-ΔKDEL-V5-His and pcDNA3/CEBPB (C) as indicated. The membranes were stained with V5 (top panel of the organelle fraction in panel A only), with PDI, CEBPA, and CEBPB Abs. The loading controls were β-actin for the cytoplasmic fraction (CF), calreticulin for the organelle fraction (OF), and lamin B for the nuclear fraction (NF). (D top panel) Western blot of whole-cell lysates from leukemic HL60 cells transfected with pcDNA3/V5-His (lane 1) or increasing amounts of pcDNA3/PDI-V5-His (lanes 2-4) and stained with PDI and CEBPA Abs. Relative densitometric quantitation of CEBPA bands indicated: 100%, 26%, 24%, and 28% (lanes 1-4). Lamin B was used as loading control. (Bottom panel) Results from 3 independent experiments of quantitative RT-PCR for CEBPA mRNA, normalized against ABL expression. (E) Western blot of whole-cell extracts from HL60 cells after transfection of unspecific siRNA as a control, of siRNA against PDI (each 900nM), and of untransfected (mock) cells, stained with PDI and CEBPA Abs. β-actin was used as loading control. (F) Western blot of whole-cell lysates from U937 cells after transfection with increasing amounts of siRNA against PDI at concentrations of 300nM, 600nM, 900nM, and of untransfected (mock) cells. Relative densitometric quantitation of CEBPA bands indicated: 18%, 46%, 92%, and 100% (lanes 1-4).