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

Figure EV5. (Related to Figs 6 and 7). PRKG2 overexpression phosphorylates Ser259 in MYRF.

Figure EV5

  1. Immunohistochemical staining with antibody to anti‐Ser259 showed that the AAV‐PRKG2 injection promoted MYRF phosphorylation in the corpus callosum (CC). Ctx: cortex, Str: striatum. Scale bar: 40 μm.
  2. The anti‐MYRF (Sigma, HAP018310) immunohistochemical staining of the corpus callosum showing no significant effect on the total MYRF expression by AAV‐PRKG2 injection. Scale bar: 100 μm.
  3. Western blotting of the corpus callosum region of PLP‐150Q/Cas9 mice indicated the expression of both Cas9 and 150Q‐HTT, compared with PLP‐150Q, transgenic Cas9 mice, and WT mice. Mutant HTT was detected by 1C2 and EM48 antibody, and Cas9 was detected by anti‐Cas9 antibody.
  4. Genome‐editing activity of PRKG2‐gRNA was tested using T7E1 assay. N2a cells were co‐transfected with PRKG2‐gRNA and Cas9 plasmids or transfected with PRKG2‐gRNA plasmid alone as control. The T7E1 assay was carried out using PCR products of genome PRKG2 DNA from transfected N2a cells. The arrow indicates cleaved PCR products by T7E1 enzyme. Control is control‐gRNA.
  5. Mutations in the CRISPR/Cas9 targeted PRKG2 gene were identified by TA cloning and subsequent sequencing.