FIG. 6.

Identification of a PIAS1 recognition motif on SATB1 and additional substrates. (A) WCEs (1,000 μg) prepared from Nalm-6 cells transiently expressing pcDNA3.1/HIS-SATB1 (WT or mutant) were harvested and coimmunoprecipitated with anti-PIAS1. Precipitates were fractionated by SDS-PAGE, electrotransferred to Immobilon-P membranes, and probed with anti-SATB1 (top panel). IgG serves as a loading control. The blot was stripped and reprobed with anti-PIAS1 (middle panel). WCEs (20 μg) were immunoblotted to anti-SATB1 (bottom panel). (B) (panel i) Shown is the region of SATB1 recognized in vivo by PIAS1. Serines, threonines, and leucines that were mutated for specific studies are shown, as is the SATB1(1-193) deletion of the LXXLL motif. (panel ii, top) Alignment of SATB1, SATB2, and CHD3-B. Symbols depict residues that are identical (asterisks), strongly conserved (diamonds), or weakly conserved (filled circles). (panel ii, bottom) Substrate motifs recognized by PIAS-SAP are aligned, and specific interaction sites are indicated at the right of the alignment; ND, not determined. The region around the LXXLL motif is predicted to be helical. (panel iii) Alignment of LXXLL motifs from protein-protein interactors (47).