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Figure 1 | BMC Biochemistry

Figure 1

From: Displacement affinity chromatography of protein phosphatase one (PP1) complexes

Figure 1

A peptide based on the PP1 interaction motif RVXF/W displaces PP1 regulatory subunits from the affinity matrix microcystin-Sepharose. Proteins extracted from isolated rat liver nuclei were incubated with the affinity (MC) or control (Con) matrix (Tris coupled). Proteins were displaced with the RPKRKRKNSRVTF SEDDEII peptide (a), while in (b), elution was performed with either RPKRKRKNSRVTF SEDDEII or GKKRVRW ADLE prior to the 3 M NaSCN elution. The control matrix is only used in the panel (a) experiment while microcystin-Sepharose is used in all others. After concentration, samples were run on 10% SDS-PAGE, blotted to nitrocellulose and membranes were probed with anti-PR65 and PP1 antibodies. In panel (c) the membrane was probed with anti-ZAP, p99 or NIPP1 antibodies [31]. To test for the salt dependence of peptide displacement, columns were eluted with peptide plus or minus NaCl as indicated and samples blotted for NIPP1 (d). To determine optimal peptide concentration for displacement from the column, the protein loaded beads were divided into 3 equal parts and eluted with 0.1, 0.5 and 2 mM peptide (e). To test for the specificity of the peptide displacement, the beads were divided into 3 equal parts and eluted with either GKKRVRW ADLE, the GKKRVRW ADLE peptide with the key interacting residues changed to A (GKKRARA ADLE), or a scrambled version of the GKKRVRW ADLE peptide (KLRGEVAKDWR) and blotted for ZAP, p99 and NIPP1. Glycogen particles were isolated from rabbit skeletal muscle and PP1GM bound to the microcystin matrix followed by peptide elution first with the GKKRARA ADLE peptide, then GKKRVRW ADLE, followed by 3 M NaSCN.

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