Rabbit liver glycogen (Sigma-Aldrich, G8876) and rabbit phosphorylase a (Sigma-Aldrich, P1261) were purchased. All assays were carried out at 30°C for 30 min if not otherwise stated. Dephosphorylation of phosphorylase a was performed at a final concentration of 2 μM in reaction buffer [50 mM tris (pH 7.8), 150 mM NaCl, and 1 mM TCEP]. To investigate the effects of the PP1 interactors GM49–86, GM64–105, NIPP1158–216, spinophilin417–602, NG1, or NG2 toward phosphorylase a dephosphorylation, the interactors were added at a 10 M excess to PP1 and incubated for 30 min at room temperature before the initiation of the assay. The final concentration of PP1 for the steady-state experiments was 0.2 μM, except for the time point experiment for which 0.04 μM PP1 was used. To test the effect of glycogen on the dephosphorylation of phosphorylase a, glycogen (4 mg/ml) was added to phosphorylase a before the addition of the GM64–237:PP1 holoenzyme. The reactions were terminated by the addition of 5× SDS loading buffer, and the samples were boiled (95°C) for 5 min. The samples were analyzed using SDS-PAGE, fixed, and stained with Pro-Q Diamond and Sypro Ruby (Thermo Fisher Scientific) to quantify the phosphor-protein (phosphorylase a) and total proteins, respectively. Gel images were captured using a ChemiDoc MP Imaging system or a Pharos FX Imager (Bio-Rad), and the densitometry of protein bands was analyzed using Image Lab 6.0 (Bio-Rad).

The GST-GYG1:GYS1 complex (final concentration of 0.1 mg/ml) in assay buffer [50 mM tris (pH 7.8), 150 mM NaCl, 1 mM TCEP, and 5% glycerol] was used in all assays. A 10 M excess of GM49–86, GM64–105, GM64–237, or spinophilin417–602 was incubated with PP1 (0.2 μM) for 30 min before the initiation of the assay. For the time point study, reactions were initiated by the addition of 0.2 μM His6-PP1α7–330 with or without GM64–237 or GM64–237 N228A. To test the effect of glycogen, glycogen (4 mg/ml) was added to GYS1 before the addition of the GM64–237:PP1 holoenzyme. The reactions were carried out at 30°C and were terminated at different time points (3, 6, 9, 12, 15, and 30 min) by the addition of 5× SDS loading buffer and boiling at 95°C for 5 min. Samples were analyzed using SDS-PAGE and stained with Sypro Ruby. Dephosphorylation of GYS1 can be readily visualized as a band shift in SDS-PAGE (30). The relative distance between the GYS1 bands relative to GST-GYG1 was used to assay the dephosphorylation of GYS1. Last, Pro-Q Diamond (Invitrogen), which stains for total phosphorylation, and mass spectrometer analysis were used to confirm the dephosphorylation states of GYS1.

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