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. 2020 Apr 9;21(6):e49783. doi: 10.15252/embr.201949783

Figure 6. The activation or overexpressing of PRKG2 promoted the phosphorylation of Ser259‐MYRF .

Figure 6

  1. Western blotting showing that the phosphorylation of transfected N‐terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8‐Br‐cGMP (10 or 100 μM) for 1 h or co‐transfected 150Q‐HTT for 24 h, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 h. trans‐nMYRF: transfected N‐terminal MYRF.
  2. Adeno‐associated virus vector expressing full‐length PRKG2 under the control of the CMV promoter.
  3. Western blotting of N2a cell line that was transfected with AAV‐PRKG2 for 48 h. The blots were probed with anti‐Ser259 (pMYRF) antibody. Note that overexpression of PRKG2 increased phosphorylation of N‐terminal MYRF (nMYRF) in a dose‐dependent manner.
  4. Stereotaxic injection of AAV‐GFP virus into the corpus callosum (CC, indicated between two dotted lines) in the mouse brain. Ctx: cortex, Str: striatum. Scale bar: 100 μm.
  5. Immunohistochemical staining with anti‐Ser259 (pMYRF) showing that AAV‐PRKG2, but not AAV‐GFP, promotes MYRF phosphorylation in the corpus callosum. Scale bar: 40 μm.
  6. Quantitative analysis of the pMYRF‐ or total MYRF‐positive cells in each field (40×). Twenty random fields in each section were examined, n = 3 mice in each group. One‐way ANOVA with Tukey's test; ***P = 8.79 × 10−5. Data are mean ± SEM.
  7. Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV‐PRKG2, but not AAV‐GFP (green), promotes MYRF phosphorylation (red) in the injected corpus callosum. Scale bar: 40 μm.
  8. Western blotting analysis of the mouse corpus callosum tissues using anti‐Ser259 (pMYRF), phosphor‐PRKG2 (pPRKG2), or phosphor‐VASP (pVASP). Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP. fMYRF: full‐length MYRF, nMYRF: N‐terminal MYRF.

Source data are available online for this figure.