Figure 2. Suppression of KE-EGFP in HEK293 cells by a short hairpin RNA targeting the coding region of the KE gene

Cells were transfected with KEpEGFP along with the control plasmid or shRNA-generating plasmid, KE-1528pH1-shRNA. The fluorescence photographs were taken at the same exposure times (140 msec) 48 h after cotransfections. A: HEK293 cells were cotransfected with KEpEGFP plasmids and control plasmids as a baseline. B: Fluorescent signals of KE-EGFP in cultured HEK293 cells were significantly reduced by cotransfection with KE-1528pH1-shRNA. C: Western blot of protein lysates of HEK293 cells from A (lane 1) and from B (lane 2) to demonstrate the reduction of KE by KE-1528pH1-shRNA is shown. Protein bands (10 μg/lane) were probed with a custom-made anti-KE antibody. Presence of KE-EGFP fusion proteins in HEK293 cells was noted after cotransfection of KEpEGFP with control plasmid in lane 1 (as indicated by KE). Significant reduction of KE-EGFP fusion proteins (as indicated by KE) was noted after cotransfection of KEpEGFP with KE-1528pH1-shRNA in lane 2. D: Northern hybridization of mRNAs from A (left lane) and B (right lane), using IR analysis by Li-Cor, is shown. Equal amounts of mRNAs were loaded in each lane as judged by the intensities of 28S RNA (as indicated by 28S). Significant reduction of IR signals of KE-EGFP mRNA (as indicated by KE) was noted after cotransfection of KEpEGFP with KE-1528pH1-shRNA in right lane.