Skip to main content
. 2011 Nov 28;287(3):2247–2256. doi: 10.1074/jbc.M111.269431

FIGURE 2.

FIGURE 2.

Sh3rf2 knockdown promotes JNK-dependent apoptosis in both proliferating and post-mitotic cells. A, upper panel, FLAG-tagged Sh3rf2 in pCMS-EGFP was expressed in HEK293 cells in the presence of either scrambled (nonsilencing) siRNA or siSh3rf2. Lysates were collected 24 h later and subjected to immunoblot for FLAG. Membranes were reprobed for GFP to confirm equal loading and transfection efficiency. In the lower panel, FLAG-tagged Sh3rf2 was co-expressed with 4-fold excess of shRNA targeting Sh3rf2 in the pU6 vector (shSh3rf2), or empty vector. Cells were collected 24 h later and immunoblotting was performed for FLAG-Sh3rf2. The membrane was reprobed with GFP to ensure equal transfection efficiency for Sh3rf2. B, neuronal PC12 cells were co-transfected with pCMS-EGFP and either scrambled siRNA or siSh3rf2. Transfected cells were counted starting 24 h after transfection (Day 1). Shown is one experiment performed in triplicate (*, p < 0.03; **, p < 0.01). The experiment was performed three times with similar results. C, an shRNA plasmid (shSh3rf2) targeting Sh3rf2 was transfected into primed PC12 cells in 4-fold excess to pCMS-EGFP ensuring all fluorescent cells express shRNA. Again EGFP+ cells were counted starting 24 h after transfection (Day 1). Shown is one experiment performed in triplicate (*, p < 0.03). The experiment was performed three times with similar results. D, C6 glioma cells were transfected with pCMS-EGFP and the indicated pre-synthesized siRNAs. Cells were fixed 24 h later, stained for eGFP, and stained with Hoechst 33342 to visualize nuclear morphology. The percentage of cells with condensed and fragmented nuclei was determined for each condition. Shown is one experiment performed in triplicate (*, p < 0.01). The experiment was performed three times with similar results. E, to confirm knockdown of endogenous Sh3rf2, C6 glioma cells were transfected with the indicated presynthesized siRNAs. Twenty hours later, cells were collected and subjected to immunoblot as indicated. The experiment was performed three times with similar results.