Fig. 1: The interaction between liprin-α1 and B56γ-PP2A requires the N-terminal SLiM of liprin-α1, and the SLiM binding pocket of B56γ.

a Lysates of COS7 and MDA-MB-231 cells (50 µg/lane) blotted with B55 or B56 isoform–specific Abs. b Lysates of COS7 cells transfected with YFP-B56α or YFP-B55α were immunoprecipitated with anti-GFP or anti-liprin-α1 Abs, and immunoblotted to reveal the indicated antigens (eliprin-α1, endogenous liprin-α1). c Immunoprecipitates with anti-FLAG from lysates of COS7 cells transfected with B56γ-FLAG were blotted for liprin-α1 and B56γ; mIgG, control non-immune mouse IgG. d Lysates from COS7 cells transfected with B56γ-GFP immunoprecipitated with anti-liprin-α1 Ab, non-immune mouse IgG (mIgG), or no Ab (–), and blotted with anti-liprin-α1 and anti-GFP Abs. e Top: alignment of N-terminus of human wildtype (liprin-α1) and mutant (liprin-α1-AA, with two amino acid substitutions within the SLiM). Bottom: lysates of COS7 cells transfected with B56γ-GFP alone, or together with either liprin-α1 -FLAG or liprin-α1-AA-FLAG, were immunoprecipitated with anti-GFP or control IgG (mIgG), and blotted to reveal siRNA resistant wildtype (WT) and mutant (AA) FLAG-liprin-α1, and B56γ-GFP. f Lysates of COS7 cells cotransfected with B56γ-GFP and either wildtype (WT) or SLiM–mutated liprin-α1-FLAG (AA, AA2, AA3) were immunoprecipitated with anti-GFP, and blotted to reveal siRNA resistant wildtype (WT) and mutant (AA, AA2, AA3) liprin-α1-FLAG, and B56γ-GFP. NT, control lysate from non-transfected cells. g Top: sequence alignment of B56α and B56γ: in yellow the mutated arginine residue: B56α-R222E and B56γ-R197E. Bottom: lysates from COS7 cells transfected with either B56γ-GFP or B56γ-R197-GFP, or cotransfected with B56γ-GFP and FLAG-tagged liprin-α1, were immunoprecipitated with anti-GFP (no Ig = control beads). Immunoprecipitates and lysates were blotted to reveal FLAG-tagged liprin-α1 (top), and B56γ-GFP (center). The top filter reprobed with anti-liprin-α1 reveals both endogenous and FLAG-liprin-α1. h The endogenous catalytic PP2A-C subunit in MDA-MB-231 cells is methylated. Filters with MDA-MB-231 cell lysates (30 µg/lane) untreated (–) or treated with NaOH (+) were incubated with Ab against the central part of the PP2A-C polypeptide recognizing both methylated and demethylated PP2A-C (total), or with two distinct Abs specific for demethylated PP2A-C. i Total (tot), cytosolic (C) and nuclear (N) fractions from different cell types were analyzed by immunolotting with the indicated Abs. j GFP-liprin-α1 interacts with the PP2A holoenzyme via B56γ. Immunoprecipitations (GFP-Trap) from 100 µg of protein lysate; lysates and unbound fractions, 10 µg protein/lane. k Mutation of the SLiM reduces the interaction of liprin-α1 with the B56γ-PP2A holoenzyme. Immunoprecipitations (GFP-Trap) from 300 µg of protein lysate; lysates and unbound fractions, 30 µg protein/lane. l Endogenous complex between liprin-α1 and PP2A in MDA-MB-231 cells. Immunoprecipitation (200 µg of protein lysate) of endogenous liprin-α1 (IP Lipr) pulls down catalytic and regulatory subunits of endogenous PP2A. IP Ctr, control immunoprecipitation with mouse Ig; 40 µg/lane of unbound fractions and lysate.