PowerPoint Presentation - Molecular and Cellular Biology

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					A                                      IP with -myc
                                                     gcn4∆
    GCN4             GCN4
                                  s. c.               h. c.
    CYC8-myc         CYC8
                                  GCN4-myc s. c. GCN4 GCN4-myc h. c. GCN4
        I P S     I P S I              P S I        P S I P S         I P S
                                                                                  Tup1p
                                                                                  myc-Cyc8p
                                                                                  myc-Gcn4p
    1    2   3    4    5      6   7    8   9   10 11 12 13 14 15 16 17 18


B
         GAL11-myc                     TUP1-myc                CYC8-myc
                          10Ala




                                                                          10Ala
                                                    10Ala
     Input




                                                            Input
                                  Input
     GST




                                                            GST
                                  GST
                 WT




                                                                     WT
                                               WT




                                                                                  myc

                                                                                  GST-Gcn4p



                                                                                  GST
     1 2     3   4    5       6    7   8   9   10 11 12 13 14       15 16 17 18

    FIG. 3. Cyc8p does not stably interact with Gcn4p in vivo or in vitro. (A) WCEs were
    prepared from yeast strains KS6 (GCN4 CYC8-myc), BY4741 (GCN4CYC8) and transformants of
    strain 249 (gcn4∆) carrying single copy (pSK1)) or high copy (pHQ1293) plasmids harboring the
    GCN4-myc allele or single copy (p1208) or high copy (pHQ1303) plasmids bearing untagged
    GCN4. The WCEs were immunoprecipitated with monoclonal c-myc antibodies. The immune
    complex were collected, resolved by SDS-PAGE, and subjected to Western blot analysis using
    anti-Tup1p antibodies or anti-myc antibodies. I, 10% of the Input WCEs; P, 100% of the pellet
    fractions from the immunoprecipitations; S, 10% of the supernatant fractions. (B) Equal amounts
    of GST, WT GST-Gcn4p(WT), and GST-Gcn4p-10Ala present in WCEs of Escherichia coli
    strains expressing these recombinant proteins were incubated with WCEs from yeast strains
    H3028 (GAL11-myc), KS1 (TUP1-myc) and KS6 (CYC8-myc). The GST proteins were
    precipitated with glutathione-Sepharose resin, resolved by SDS-PAGE, and detected by Western
    blot analysis using anti-myc antibodies. The bottom section of B panel shows Ponceau S staining
    of the Western blots to reveal the amounts of GST proteins precipitated in the assays. Input lanes
    contained 1% of the WCEs used in the pull down assays.




                                                                                                         Fig. S3

				
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