Fig 1: PLPP/CIN-mediated NF2 dephosphorylation.a–e In vitro assay using recombinant proteins. a Representative western blot data demonstrating PLPP/CIN-mediated NF2 dephosphorylation. b, c Quantification of NF2-S10 (b) and -518 (c) phosphorylation levels based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (*p < 0.05 vs. the presence of PLPP/CIN and PKAc; n = 7). d Representative co-immunoprecipitation data demonstrating PLPP/CIN-NF2 and -PKAc bindings. e Quantification of PLPP/CIN-NF2 and -PKAc bindings. Open circles indicate each individual value. Horizontal bars indicate mean value (*p < 0.05 vs. the presence of PLPP/CIN and PKAc; n = 7). f, g Effects of KA on PLPP/CIN-NF2 and -PKAc bindings in WT and PLPP/CINTg mice in vivo. f Representative co-immunoprecipitation data demonstrating PLPP/CIN-PKAc and NF2 bindings. g Quantification of PLPP/CIN-PKAc and -NF2 bindings. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#p < 0.05 vs. WT and control animals, respectively; n = 7).
Fig 2: Effects of NF2 knockdown on CA3 neuronal death in WT, PLPP/CINTg, and PLPP/CIN-/ mice following KA injection and the role of PLPP/CIN in NF2-S10 and Mdm2-S166 dephosphorylation.a Representative photos of FJB-positive degenerating neurons in the CA1 region. b Quantification of the number of FJB-positive neurons in response to KA. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#p < 0.05 vs. WT and control siRNA-treated animals; n = 7, respectively). c Scheme of the role of PLPP/CIN in NF2-S10 and Mdm2-S166 dephosphorylation. PLPP/CIN-mediated NF2 dephosphorylation decreases F-actin stability and increases NMDAR-PSD95 co-assembly by eliminating the local F-actin barrier. PLPP/CIN also inhibits Mdm2 activity as an E3 ubiquitin ligase for PSD95 by enhancing its S166 dephosphorylation and NF2-mediated Mdm2 degradation.
Fig 3: Effects of PLPP/CIN deletion on seizure activity, NF2 phosphorylation, and PLPP/CIN-NF2 binding in response to KA.a Representative EEG traces and frequency-power spectral temporal maps in response to KA. b Quantification of total EEG power (seizure intensity) in response to KA (#p < 0.05 vs. WT animals; n = 7, respectively). c Representative western blots of PLPP/CIN, NF2, pNF2-S10, pNF2-S518, PKAc, and pPKAc in WT and and PLPP/CINTg mice in vivo. d–h Quantification of NF2, pNF2-S10, pNF2-S518, PKAc, and pPKAc levels. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#p < 0.05 vs. WT and control animals, respectively; n = 7). i, j Effects of KA on PLPP/CIN-NF2 and -PKAc bindings in WT and PLPP/CIN-/- mice in vivo. i Representative co-immunoprecipitation data demonstrating PLPP/CIN-PKAc and NF2 bindings. j Quantification of PLPP/CIN-PKAc and -NF2 bindings. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#p < 0.05 vs. WT and control animals, respectively; n = 7).
Fig 4: Effects of PLPP/CIN over-expression on NF2 phosphorylation and seizure activity in response to KA.a Representative western blots of PLPP/CIN, NF2, pNF2-S10, pNF2-S518, PKAc, and pPKAc in WT and and PLPP/CINTg mice in vivo. b–f Quantification of NF2, pNF2-S10, pNF2-S518, PKAc, and pPKAc levels. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#p < 0.05 vs. WT and control animals, respectively; n = 7). g Representative EEG traces and frequency–power spectral temporal maps in response to KA. h Quantification of total EEG power (seizure intensity) in response to KA (#p < 0.05 vs. WT animals; n = 7, respectively).
Fig 5: Effects of NF2 knockdown on protein and phosphorylation levels of NF2, Mdm2, and PSD95 in PLPP/CIN−/− mice following KA injection, and linear regression analysis between NF2 and Mdm2/PSD95 levels.a Representative western blots of NF2, pNF2-S10, pNF2-S518, Mdm2, pMdm2-S166, and PSD95. b–g Quantification of NF2, pNF2-S10, pNF2-S518, Mdm2, pMdm2-S166, and PSD95 levels based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (*,#,$p < 0.05 vs. WT, control siRNA- and control (non-KA-treated) animals, respectively; n = 7). h, i Linear regression analysis between NF2 and Mdm2/PSD95 levels within control siRNA- and NF2 siRNA-treated groups of WT and PLPP/CINTg mice following KA injection.
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