Fig 1: The activation or overexpressing of PRKG2 promoted the phosphorylation of Ser259-MYRF 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.Adeno-associated virus vector expressing full-length PRKG2 under the control of the CMV promoter.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.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.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.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.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.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.
Fig 2: (Related to Figs 6 and 7). PRKG2 overexpression phosphorylates Ser259 in MYRF 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.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.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.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.Mutations in the CRISPR/Cas9 targeted PRKG2 gene were identified by TA cloning and subsequent sequencing.
Fig 3: Knocking down PRKG2 alleviated demyelination and decreased phosphorylation of MYRF in PLP-150Q/Cas9 mice Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 µm.Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2, or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from three independent experiments were presented under the blots. Student's t-test; pMYRF: ***P = 0.00026; MBP; **P = 0.0036. Data are presented as mean ± SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 µm.Quantitative analysis of the pMYRF-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 = 0.00022. Data are mean ± SEM.Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 µm.Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40×) in each section were examined. n = 3 mice in each group. One-way ANOVA with Tukey's test; ***P = 4.43 × 10-5. Data are mean ± SEM.A proposed model for the protective effect of LAQ in HD mice. LAQ increases myelin protein expression by reducing the expression of PRKG2, leading to reduced phosphorylation of MYRF and its dissociation from mutant HTT and therefore increasing its transcriptional activity to express myelin-associated genes. Source data are available online for this figure.
Fig 4: LAQ suppresses PRKG2 gene transcript expression via AhR negative regulation The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from three independent experiments. One-way ANOVA with Tukey's test; ***P < 0.001. Data are mean ± SEM.The treatment of LAQ (5 or 10 µM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from three independent experiments. One-way ANOVA with Tukey's test. ***P < 0.001. Data are mean ± SEM.The putative cis-elements (in box and sequences below) in the PRKG2 promoter were determined by TFSEARCH, ConTra, and ALGGEN program analyses. Six AhR transcriptional factor binding sites were predicted according to the conserved sequence of “CACGC” or “GCGTG”.The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n = 3 mice in each group. Student's t-test; Cyp1a1: ***P = 0.00042; Ugt1a6a; ***P = 0.00057. Data are mean ± SEM.The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 µM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANOVA with Tukey's test. ***P < 0.001. Data are mean ± SEM.The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 µM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 h. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 µM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 h. The results were obtained from three independent experiments. One-way ANOVA with Tukey's test. LAQ: ***P = 0.00065; 2,3,7,8-TCDD: ***P = 0.00014. Data are mean ± SEM. Source data are available online for this figure.
Fig 5: (Related to Figs 4 and 5). Mutant HTT promotes PRKG2 gene transcript expression N2a cells transfected with 23Q-HTT or 150Q-HTT were examined by the antibody of PRKG2 or phosphor-VASP (pVASP). Note that both the expression of PRKG2 and pVASP was increased by 150Q-HTT. GAPDH served as control. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from three independent experiments were presented on the right. One-way ANOVA followed with Tukey's test. PRKG2: ***P = 0.00032; p-VASP: ***P = 0.00059. Data are mean ± SEM.The co-transfection of 150Q-HTT with core promoter of PRKG2 (-472/+131) displayed an obvious induction of the transcription activity from three independent experiments, compared with the 23Q-HTT or Mock control. One-way ANOVA followed with Tukey's test. ***P < 0.001. Data are mean ± SEM.Western blotting of transfected N2a cells showing that AhR siRNA, but not the scrambled control siRNA, treatment for 72 h inhibited AhR protein expression. The blots were probed with anti-AhR antibody.
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