Con A–Sepharose (GE Healthcare) beads were used to assay glycogen binding. Fresh Con A beads were washed in Con A buffer [67 mM Hepes (pH 6.8), 0.2 mM CaCl2, 10 mM MgCl2, 500 mM NaCl, and 4 mM DTT] and incubated with rabbit liver glycogen (50 mg/ml; Sigma-Aldrich, G-8876, 1:1 volume) at 4°C for 1 hour. Glycogen-conjugated Con A beads were washed three times with Con A buffer and resuspended as 50% slurry. Con A–glycogen Sepharose beads (30 μl) were incubated with 15 μg of phosphorylase a, GM64–237, and PP1α7–330 alone and in various combinations thereof in a total volume of 250 μl for 1 hour at 4°C under gentle mixing. The beads were washed three times with Con A buffer (750 μl) and recovered by centrifugation (2000g, 1 min). The supernatant was removed using gel-loading tips, leaving behind the beads that were then incubated with 25 μl of 1 M α-d-methylglucoside (in Con A buffer) to elute all proteins bound to glycogen. Eluate (10 μl) was carefully transferred to new tubes to avoid contamination with residual beads and analyzed by SDS-PAGE. SDS-PAGE was stained using Sypro Ruby (Invitrogen) for visualization of total proteins.

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