Figure 3
PKA phosphorylates SOX11 in serine 133. (a) Mass Spectrometry analysis of the in vitro phosphorylation assay. The table reports the spectral data for the phosphopeptide corresponding to Sox11 pS133/137, including the number of spectra with a peptide probability > 50% (Scaffold); the Mascot ion, identity and delta scores; the type of residue modifications, the theoretical (actual) as well as the observed mass; the peptide charge; the delta mass in Dalton and PPM; the retention time, the total ion count (TIC), the start and stop positions within the murine SOX11 amino acid sequence. (b) Comparison of the sequence around S133 and S137 with pkaPS. The table reports that PKA is predicted to phosphorylate S133 with score 0.29 but not S137 (score -1.41). Immunofluorescent analysis and line intensity plots of the subcellular localization (c–c’) of SOX11WT in HEK293T cells overexpressing pCAG–Sox11WT–IRES–GFP, (d–d’) of SOX11S133NON in HEK293T cells overexpressing pCAG–Sox11S133NON–IRES–GFP, and (e-e’) of SOX11S133MIMIC in HEK293T cells overexpressing pCAG–Sox11S133MIMIC–IRES–GFP. SOX11 (in red) and DAPI (in blue). Scale bars = 20 μm. (c’–e’) Representative line intensity plots of HEK293T cells transfected with SOX11 wildtype and mutants. The blue line represents the intensity of DAPI, the red line represents the intensity of SOX11 along a cross-section of a cell. The left intensity plot shows a cell with nuclear SOX11 localization (note the overlap between the DAPI and the SOX11 signal), while the right intensity plot shows a cell with nuclear and cytoplasmic SOX11 localization (note that the SOX11 signal extends beyond the DAPI signal).
