Figure 5.

Selective knock-down of GODZ by GODZ-specific shRNA. a, Analysis of the target specificity of shRNA constructs. A control vector encoding a mutated GODZ shRNA target sequence or one of two GODZ-specific shRNA constructs (GODZ shRNA 1, 2) were cotransfected with either FL-GODZ or FL-SERZ-β or FL-DHHC15 into HEK293T cells, and the cell extracts were analyzed by Western blot using mAb anti-FLAG. The blots were stripped and reprobed with anti-tubulin antibody as a loading control. Note the dramatic reduction in GODZ in cells cotransfected with GODZ shRNA 1 or 2, whereas SERZ-β and DHHC15 expression was unaffected under the same conditions. No effect on GODZ, SERZ-β, or DHHC15 expression was observed on cotransfection with the mutant shRNA control vector. b, Quantitation of GODZ shRNA-mediated effects on GODZ, SERZ-β, and DHHC15 expression in transfected HEK293T cells by flow cytometry (see Materials and Methods). The cells were transfected with GFP-GODZ, GFP-SERZ-β, or GFP-DHHC15 together with GODZ shRNA 1, GODZ shRNA 2, or control shRNA vector. Note the drastic reduction in GFP-GODZ expression by cotransfected GODZ shRNA1 (15.15 ± 0.91% of control shRNA; n = 6; p < 0.001) or GODZ shRNA2 (11.48 ± 1.48% of control; n = 6; p < 0.001). In contrast, neither of the two GODZ shRNA constructs had an effect on expression of GFP-SERZ-β (GODZ shRNA1, 109 ± 8.3% of control shRNA, n = 12, p > 0.05; GODZ shRNA2, 113 ± 8.7%, n = 12, p > 0.05) or GFP-DHHC15 (GODZ shRNA1, 95 ± 3.7% of control shRNA, n = 5, p > 0.05; GODZ shRNA2, 92 ± 5.0%, n = 6, p > 0.05).