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. Author manuscript; available in PMC: 2008 Jun 3.
Published in final edited form as: J Biol Chem. 2007 Jun 12;282(31):22335–22343. doi: 10.1074/jbc.M701477200

FIGURE 3. PIAS3 is an ATF1-binding protein.

FIGURE 3

A, yeast two-hybrid analysis of ATF1-PIAS3 interaction. PJ69-4A yeast cells were transformed with pGBD and pAct2 (-/-), pGBDATF1 and pAct2 (ATF1/-), pGBDATF1 and pAct2PIAS3 (ATF1/PIAS3), and pGBD and pAct2PIAS3 (-/PIAS3), and transformed colonies were tested for their growth on SD-adenine deficient agar plate. pGBDATF1 is a human full-length ATF1, and pAct2 PIAS3 is a mouse C-terminal PIAS3 isolated from pAct2 NIH3T3 cDNA library. B,1 μg of pCMVHA-hATF1 and 9 μg of pCMVFLAG-hPIAS3 were transfected into 293 cells, and cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody followed by Western blotting (WB) with anti-HA antibody (top panel), or whole cell lysates were subjected to Western blotting with anti-HA antibody (middle panel) or anti-FLAG antibody (bottom panel). pCMVHA-ATF1 transfected cell lysate was simultaneously loaded on the IP/WB gel to identify the HA-ATF1 band. A representative of four independent experiments is shown. C, 800 μg of 293 whole cell lysates were immunoprecipitated with 1 μg of rabbit IgG or anti-PIAS3 antibody, followed by Western blotting with anti-ATF1 family antibody. 50 μg of whole cell lysates were used for Western blots of ATF1/CREB and PIAS3 to confirm the positions of these proteins in the SDS-PAGE gel. A representative of three independent experiments is shown.