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. 2015 Jun 12;290(31):19044–19054. doi: 10.1074/jbc.M115.637660

FIGURE 2.

FIGURE 2.

Characterization of the GPS2 nuclear localization domain. A, schematic of GPS2 structure, including the coiled coil (CC) domain, and constructs utilized in Fig. 1 with a summary of the results in terms of nuclear localization (NL) of the different mutants that led to the identification of aa 40–60 as the minimal NLD. B, GST pulldown assays confirming that GPS2 Δ60–154 interacts with TBL1 Nt as well as GPS2 wild-type. The interaction with GST-TBL1 Ct was measured as a negative control. C, Western blot for HA-GPS2 in HeLa NEs and CEs showing that adding aa 1–60 to the GPS2 Ct domain (GPS2 Δ60–154) rescues GPS2 expression and targeting to the nucleus. D, site-directed mutagenesis of a putative NLS (K52A/K53A) within the NLD does not impair GPS2 nuclear localization. E, coimmunoprecipitation (IP) of TBL1 and GPS2 from HeLa nuclear extracts upon transfection with control siRNA (sictl) or siUbc9 and HA-GPS2 showing that inhibiting sumoylation does not impair GPS2 nuclear expression or disrupt the interaction between GPS2 and TBL1. F, GST pulldown assay testing the direct interaction between GST-TBL1 Nt and recombinant GPS2 (wild-type, K45R, K71R, or double K45R/K71R mutants).