蛋白質(zhì)相互作用研究方法

1、會(huì)計(jì)學(xué)1蛋白質(zhì)相互作用研究方法蛋白質(zhì)相互作用研究方法UASs, upstream activating sequencesAD, activating domainBD, binding domainFigure 1. The CysSer mutation of HPO destroying its enzyme activity disturbed neither HPO dimerization nor HPO-JAB1 interaction in vivo. C) The indication of yeast cells containing vectors in each pie

2、 slice of the culture plate. D) Growth of transformants coexpressing HPO and JAB1 on selective leu/trpmedium. E) Growth of transfected yeast cells on leu/trp/his medium. 30 kD15 kDGST-JAB1 GST Coomasssie blue stainFigure 2. JAB1 binds to rHPO expressed in E.coli in vitro: rHPO were applied to column

3、s of glutathione beads bearing GST or GST-JAB1.The bound rHPO was eluted together with GST-JAB1, was separated by SDS-PAGE, transferred to PVDF, and detected by anti-HPO antibody. The GST and GST-JAB1 were visualized by Coomasssie blue staining. Blot: rHPOGST + rHPOGST-JAB1 + rHPOrHPOFigure 3. The C

4、ysSer mutation of HPO destroying its enzyme activity disturbed neither HPO dimerization nor HPO-JAB1 interaction in vivo. COS 7 cells were cotransfected with JAB1 and either Myc-tagged HPOs (wild type or mutants) or Myc control vector. The cells lysates were incubated with a rabbit anti-JAB1 antibod

5、y then with protein A/GAgarose. The immuno-complex was resolved on 15% SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody. As an indication of the relative expression level for Myc-HPO, some of the total cell lysate used in immuno-precipitation was loaded onto lanes 8 (pCMV-Myc-HPOwt) an

6、d 9 (pCMV-Myc). Lanes 2 to 7 are from cells expressing JAB1/Myc and JAB1/Myc-HPO (HPOwt, HPOC67S, HPOC70S, HPOC67S/C70S and HPOC90S), respectively. Lane 1 is a control for lane 3 with rabbit mock antibody (normal IgG). A duplicate blot was also probed with anti-JAB1 antibody to monitor the amounts o

7、f JAB1 protein (bottom). IP, immuno-precipitation; IB, immuno-blotting. Fig.21 Cellular localization of HPO and JAB1. COS 7 cells were tranfected with GFP-HPO or RFP-JAB1 constructs, respectively. The nuclei were stained by Hoechst 33342 (blue). All cell samples were visualized by confocal microscop

8、y (Leica). Fig.22 Cellular colocalization of JAB1 with HPO. COS 7 cells were cotransfected with RFP-JAB1 and GFP-HPO constructs. The nuclei were stained by Hoechst 33342 (blue). Arrowheads indicate the field of colocalization. All cell samples were visualized by confocal microscopy (Leica).Fig.23 En

9、dogenous JAB1 colocalizes with endogenous HPO in HepG2 cells. HepG2 cells were fixed in 30% paraformaldehyde, permeabilized in 0.5% Triton X-100, stained with anti-HPO rabbit polyclonal and anti-JAB1 mouse monoclonal antibodies, and incubated with fluorescin (FITC)-linked anti-mouse and Texas red (T

10、R)-linked anti-rabbit IgG. All cell samples were viewed by confocal fluorescensemicroscope (Leica). Figure 3. The CysSer mutation of HPO destroying its enzyme activity disturbed neither HPO dimerization nor HPO-JAB1 interaction in vivo. COS 7 cells were cotransfected with JAB1 and either Myc-tagged

11、HPOs (wild type or mutants) or Myc control vector. The cells lysates were incubated with a rabbit anti-JAB1 antibody then with protein A/GAgarose. The immuno-complex was resolved on 15% SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody. As an indication of the relative expression level for Myc-HPO, some of the total cell lysate used in immuno-precipitation was loaded onto lanes 8 (pCMV-Myc-HPOwt) and 9 (pCMV-Myc). Lanes 2 to 7 are from cells expressing JAB1/Myc and JAB1/Myc-HPO (HPOwt, HPOC67S, HPOC70S, HPOC67S/C70S and HPOC90S), respectively. Lane 1 is a control for lane 3 wit