1. PP1 preferentially binds nonphosphorylated RV[S/T]F motifs in vitro.
PP1 preferentially binds nonphosphorylated RV[S/T]F motifs in vitro. (A) Phosphorylated (P) or nonphosphorylated (nonP) versions of RV[S/T]F-containing peptides from the indicated proteins were spotted onto nitrocellulose membranes in the indicated amounts. The membranes were overlaid with a mixture of recombinant human PP1 and probed with a PP1-specific antibody. A peptide derived from the PP1-binding protein YLP motif–containing protein 1 (YLPM1) (RVRW) was used as a positive control, and the nonphosphorylable mutant version of this peptide (RARA) was used as the negative control. (B) Activated 5-carboxypentyl–Sepharose 4B N-hydroxysuccinimide ester (CH-Sepharose) beads were coupled to the phosphorylated or nonphosphorylated versions of the indicated RV[S/T]F peptides and incubated with clarified HeLa whole-cell extracts. After washing, proteins bound to the beads were eluted with SDS sample buffer, run on SDS–polyacrylamide gel electrophoresis (PAGE), and immunoblotted for PP1. The position of the 35-kDa mass marker is indicated. Peptides derived from the indicated proteins and sequences are shown in table S1. The data represent one of three independent experiments for both (A) and (B).
Isha Nasa et al., Sci. Signal. 2018;11:eaai8669
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