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Control

Control. Nodal. ALK7-ca.

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Control

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  1. Control Nodal ALK7-ca Sl Fig. 1. Identification of potential Nodal/ALK7 target genes using genearray. Total RNA was extracted from IOSE cells transfected with control vector, Nodal, or ALK7-ca and subjected to reverse transcribed. The cDNA samples were then labeled with biotin and hybridized to specific cDNA on the membrane (Human Cell Cycle Gene Array from SupperArray). Large arrow indicates cyclin G2 and small arrow indicates Skp1. Table listed genes that are up- or down-regulated by Nodal and ALK7-ca.

  2. V5 His Stop 1 1 1 1 1 2 2 2 2 2 12h 24h 48h Post-transfection B V5 His Stop 0 5 10 15 0 5 10 15 0 5 10 15 mg CCNG2-V5 V5 myc His His Stop Stop b-actin 116 a b c d e CCNG2-V5 82 64 GFP Stop 48 37 b-actin 26 19 3xFlag Stop Kpn I Xba I Age I A pcDNA3.1-CCNG2-V5 Kpn I Age I pcDNA4-CCNG2-V5 Kpn I Sac II pcDNA3-CCNG2-V5-His Kpn I Sac II pcDNA4-CCNG2-myc-His EcoR I Sac II pCCNG2-GFP EcoR I BamH I p3xFLAG-CCNG2 OV2008 IOSE C Sl Fig. 2. Generation of cyclin G2 expression constructs and detection of cyclin G2 protein. A) Generation of human CCNG2 plasmids. These plasmids contain the fusion tags either at N-terminus or C-terminus. Arrow indicates a start site of translation. B) Detection of exogenous CG2 in a dose and time-dependent manner. IOSE397 and OV2008 cells were transiently transfected with CG2-V5 plasmid. CG2 fusion protein was detected using a V5 antibody. β-actin was used as control for the equal loading. C) Representative Western blots probed with different cyclin G2 antibodies. OV2008 cells were transiently transfected with an empty vector (1) or CCNG2-V5 (2). Proteins were extracted at 6 hours post-transfection and subjected to SDS-PAGE. Blots were probed with anti-V5 from Invitrogen (a), anti-CCNG2 from Abcam (b), anti-CCNG2 from Epitomics (c); anti-CCNG2 from Santa Cruz (d), and anti-CCNG2 from Abnova (e). Only the antibody from Santa Cruz appeared to recognize cyclin G2.

  3. Xu et al., Fig. Sl3 A DAPI CCNG2 pcDNA Merge DAPI CCNG2 Skp2 Merge DAPI CCNG2 ALK7-ca Merge B DAPI CCNG2 Skp2 Merge DAPI CCNG2 Skp2 Merge Sl Fig. 3. Effect of Skp2 on cyclin G2 expression as detected by immunofluorescent staining. A) Cells were co-transfected with cyclin G2 and control vector (pcDNA4, upper panel), Skp2 (middle panel), or ALK7-ca (lower panel). In cells expressing Skp2, cyclin G2 level was low whereas in cells expressing ALK7-ca, strong cyclin G2 signal was detected. B) Cells were transfected with cyclin G2 and control-siRNA (upper panel) or Skp2-siRNA (lower panel). Strong cyclin G2 signals were observed in the presence of Skp2-siRNA compared to control-siRNA. Scale bar: 100mm.

  4. OV-GFP OV-siCCNG2 GFP Phase Xu et al., Fig. Sl4 Sl Fig. 4. Generation of OV2008 stable cell lines expressing control vector (OV-GFP) or cyclin G2 siRNA (OV-CCNG2siRNA). GFP-positive clones were selected by fluorescent microscopy.

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