Fig. 2: CHIP is phosphorylated by PKG at Serine 20.

a Immunoprecipitation of Myc-tagged CHIP with PKG1α. Lanes: 1-input, 2 negative control, 3-IP signal after three bead washes. Experiment replicated ×3. b Mass spectroscopy detects increased Chip-S20 (mouse sequence) phosphorylation with acute PKG activation. The lower schematic shows the sequence and fragmentation matching the spectra (above). The M2S spectra has red annotations for the assigned ion fragments, with three fragment ions localizing phosphorylation at S20 (b8 and y11 indicate it occurs on the N-terminal sequence of LGTGGGGS, b7 indicates the sequence LGTGGGG does not contain the site. Study performed with 3 biological replicates. c CHIP peptide sequence from start of N-terminus shows high level of conservation including three serines (S19, S23, S25 for human; S20, S24, S26 for mouse and rat). Lysine 30 (K30 human, K31 mouse) is also nearby, highly conserved, and known to be critical for CHIP-chaperone binding. d Mass Spec detection of serine phosphorylation of recombinant human CHIP peptide fragment containing residues 13–30 (Uniprot Q9UNE7). Only S20 phosphorylation is detected with WT CHIP. If S20A mutation is expressed, some reduced phosphorylation is observed at S24 and S26. All biological replicates shown in figure, truncated violin plot, median and 25/75%; unpaired t-test, *p = 0.01 vs CHIP-WT. e Recombinant PKG1α/CHIP assay shows pS20 signal by immunoblot for full length WT but not S20A mutant. Example of three biological replicates. f Phospho S20 antibody detects CHIP phosphorylation at S20 in rat myocytes exposed to cGMP or PDE5A inhibition, and both are blocked by concomitant PKG inhibition with DT3 (1 μM). Gel repeated ×3, with 3–4 biological replicates. Summary analysis in Supplementary Fig. 2a.