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. 2007 Feb 5;27(7):2661–2675. doi: 10.1128/MCB.01098-06

FIG. 4.

FIG. 4.

PIAS family members (but not Pc2) are E3 ligases for HIC1 in vivo. (A) Cos-7 cells were transfected with 1.25 μg of FLAG-HIC1 and 1.25 μg His-SUMO-1 expression vectors (lane 1) or 125 ng of His-SUMO-1 expression vector alone (lane 2) or together with the expression vectors for PIAS1, PIASγ, PIASXβ, and PIASXα (lanes 3 to 6). Forty-eight hours after transfection, cell lysates were subjected to nickel-agarose precipitation, separated by SDS-PAGE, and immunoblotted with the anti-FLAG monoclonal antibody. The lack of HIC1 SUMOylation in the presence of PIASγ and PIASXβ (lanes 4 and 5) is due to the fact that these overexpressed E3 ligases conjugate the limiting amount of SUMO-1 onto their own endogenous substrates, thus precluding the SUMOylation of HIC1. (B) Cos-7 cells were transfected with 1.25 μg of FLAG-HIC1 expression vector alone (lane 1) with 1.25 μg His-SUMO-1 expression vector (lane 2) or 125 ng of His-SUMO-1 expression vector together with the expression vectors for PIAS1Xα or Pc2 (lanes 3 and 4). Forty-eight hours after transfection, cells were lysed in buffer (RIPA) containing 20 mM NEM. Lysates were subjected to immunoprecipitation (IP) with the polyclonal anti-HIC1 antibody, separated by SDS-PAGE, and immunoblotted with the anti-FLAG monoclonal antibody. WB, Western blot.

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