Flag-Tag Antibody - #T0003

Fig. 4 |DRD2 overexpression inhibited the ubiquitin-dependent degradation of HIF1α. N, Effect of DRD2 on the content of ubiquitin binding to HIF1α. DRD2-overexpressing cells treated with MG132, and cellular extracts were prepared for co-IP assay with anti–HIF1α antibody, followed by Western blotting detection of ubiquitin. siRNA, small interfering RNA. ns, nonsignificant; *, P < 0.05; **, P < 0.01

Figure 6.| FBXW7-185aa, Encoded by circFBXW7, Inhibited the Proliferation and Metastasis of TNBC Cells through Downregulating c-Myc Expression.s. (H)FBXW7-185aa-FLAG-overexpressing BT549 cells were cotransfected with sh-FBXW7 or USP28 vectors. The FBXW7-185aaFLAG, FBXW7, USP28, and c-Myc expression levels were quantified by western blot analysis.

Supplemental Figure 1| Pdx1 flag is expressed in most P0.5 and 6W Ngn3-Cre;CAT-Pdx1 islet cells. Flag staining (red) was observed in most of Ngn3-Cre;CAT-Pdx1 insulin + (blue) and glucagon + (green) cells at P0.5 and 6W; the Crepenetrance rate was a maintained at approximately 95%. Scale bars: 50 mm.

Fig. 6.| SIRT5 promotes ccRCC cell proliferation by interacting with and desuccinylating SDHA. (A-B) ACHN cells were transfected with scramble shRNA or SIRT5 shRNA plasmid, SDHA was precipitated and its succinylation level was probed by anti-pan-succinylation antibody. SIRT5 silencing led to hypersuccinylation of SDHA in ccRCC cells and increased activity of SDHA. (C-E) SIRT5 silencing decreased cellular NADPH/NADP+and GSH/GSSG ratio and increased cellular ROS level. (F) Co-IP of SIRT5 and FLAG-tagged WT SDHA in HEK293A cells revealed SIRT5 coimmunoprecipitated with SDHA. (G-J) SIRT5 silencing repressed ACHN and 769-P cell proliferation and colony formation. Methods followed Fig. 5G-J.

Fig. 5Npn-2 controls axon collateral formation. A Npn-2 knockdown by shNpn-2 validated in primary neuron cultures by western blot using an antibody for rat Npn-2. B Quantitation of Npn-2 in A, n = 3, ***P = 0.0003. C hNpn-2 expression validated by western blot. Expression of hNpn-2 in neurons was detected by western blot using an anti-Flag antibody, showing that the expression of human full-length Npn-2 cannot be affected by the rat shNpn-2 used. D Quantitation of Npn-2 in C, n = 3, P > 0.05. E Neonatal rat primary hippocampal neurons were transfected with shCtrl, shNpn-2, or shNpn-2 plus hNpn-2 plasmids at DIV 0 and treated with either control medium or Sema3F (5 nM) at 48 h after transfection for 24 h. Axons and their collaterals were detected by immunofluorescent staining with GFP and Tau-1. Arrows indicate main axons and stars indicate axon collaterals. Scale bar, 40 μm. F, G Quantitation of axon collateral number and length in E. Sema3F treatment reduced the number and length of axon collaterals in neurons transfected with shCtrl but not in neurons with shNpn-2 transfection, which was rescued by hNpn-2 co-transfection. *P < 0.05, **P < 0.01. H Quantitation of main axon length in E. Main axon elongation in neurons transfected with shCtrl was inhibited upon Sema3F treatment, but not in neurons with shNpn-2 transfection, which was rescued by hNpn-2 expression. **P < 0.01, ***P < 0.001. One-way ANOVA, post hoc Tukey test. Error bars represent SEM

FIGURE 1 Gankyrin promotes migration and invasion in OS in vitro. A, Induction of gankyrin in U2OS and MG63 cells was confirmed by Western blot analysis. B, Knockdown of gankyrin in U2OS and MG63 cells was confirmed via Western blot analysis. C, Induction of gankyrin significantly increased OS cell migration ability, as shown by wound healing assays. *P < .05; **P < .01. D, Up‐regulated gankyrin remarkably promoted OS cell invasion, as shown by Transwell invasion assays. *P < .05. E, Knockdown of gankyrin reduced OS cell migration ability. **P < .01. F, Knockdown of gankyrin inhibited OS cell invasion. **P < .01; ***P < .001