s186  (Almac Inc)

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A ) Representative western blots from total brain lysate of APP/PS1 mice or their WT littermates at 8 weeks of age (left). Quantification of Mdm2 ( P = 0.1442), phosphor (p)-Mdm2 at S166 (** P = 0.0019), and p-Mdm2 at S186 ( P = 0.9126) (right). n = 12–14 for WT and APP/PS1 mice, respectively. ( B ) Representative western blots from WT primary cortical neuron cultures treated with amyloid-beta 1–42 (Aβ42; 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. (Left) Quantification of Mdm2 ( P = 0.1169), phosphor (p)-Mdm2 at S166 (* P = 0.0363), and p-Mdm2 at S186 ( P = 0.6125) (right). For Mdm2 and p-Mdm2 at S166, n = 13 from seven independent cultures for both Scr and Aβ42 groups. For p-Mdm2 at S186 n = 7 from three independent cultures. ( C ) Representative western blots from WT primary cortical neuron cultures treated with MK-2206 for 30 min, followed by Aβ42 or Scr (1 µM) for 2 h at DIV 12–14 (left). Quantification is performed by first normalizing p-Mdm2 at S166 to total Mdm2 levels, followed by normalizing Aβ42 group to Scr group within DMSO or MK-2206 group. n = 11 from six independent cultures for both Scr and Aβ42 groups. ( D ) Representative western blots from total brain lysate of APP/PS1 mice or their WT littermates at 8 weeks of age (left). Quantification of Akt ( P = 0.8666), p-Akt at S473 ( P = 0.6823), and p-Akt at T308 ( P = 0.4952) (right). For Akt and p-Akt at S473, n = 9 for WT and APP/PS1 mice. For p-Akt at T308, n = 12 for WT and APP/PS1 mice. ( E ) Representative western blots from WT primary cortical neuron cultures treated with Aβ42 or Scr (1 µM) for 2 h at DIV 12–14 (left) Quantification of Akt ( P = 0.8785), p-Akt at S473 ( P = 0.6669), and p-Akt at T308 ( P = 0.6716) (right). For Akt and p-Akt at S473, n = 8 from two independent cultures for Scr and Aβ42 groups. For p-Akt at T308, n = 14 from three independent cultures for Scr and Aβ42 groups. Data Information: Significance was determined by Student’s t test ( D ) or Mann–Whitney U test ( A , B , C , E ). Data are represented as mean ± SEM with * P < 0.05, ** P < 0.01, **** P < 0.0001, ns non-significant. .

Article Snippet: Mouse anti-PSD-95 antibody (#sc32290), mouse anti-phospho-Mdm2-S166 antibody (#sc-965), and mouse anti-phospho-Mdm2-S186 antibody (#sc-53368) are from Santa Cruz Biotechnology.

Techniques: Western Blot, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: Quantification of interaction between eEF1α and Mdm2 after co-immunoprecipitation and representative western blots using lysates from WT primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled Aβ peptide (Scr, 1 µM) for 2 h at DIV 12–14. n = 10 at least from three independent cultures for both Scr and Aβ42 groups. Significance was determined by Student’s t test ( P = 0.4605). Data are represented as mean ± SEM with ns: non-significant. .

Article Snippet: Mouse anti-PSD-95 antibody (#sc32290), mouse anti-phospho-Mdm2-S166 antibody (#sc-965), and mouse anti-phospho-Mdm2-S186 antibody (#sc-53368) are from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A ) Quantification of p53 ( P = 0.7976) and representative western blots from total brain lysate of APP/PS1 mice or their WT littermates at 8 weeks of age. n = 11 and 10 for WT and APP/PS1 mice, respectively. ( B ) Quantification of p53 ( P = 0.6118) and representative western blots from WT primary cortical neuron cultures treated with amyloid-beta 1–42 (Aβ42; 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. n = 8 from two independent cultures for Ctrl and Aβ42 groups. ( C ) Quantification of PSD-95 (* P = 0.0477) and representative western blots from total brain lysate of APP/PS1 mice or their WT littermates at 8 weeks of age. n = 13 and 14 for WT and APP/PS1 mice, respectively. ( D ) Quantification of PSD-95 (*** P = 0.0001) and representative western blots from WT primary cortical neuron cultures treated with Aβ42 (1 µM) or Scr (1 µM) for 2 h at DIV 12–14. n = 5 and 4 from three independent cultures for Scr and Aβ42 groups, respectively. ( E ) Quantification of interaction between PSD-95 and Mdm2 ( P = 0.0084) after co-immunoprecipitation and representative western blots using lysates from APP/PS1 mice or their WT littermates at 8 weeks of age. n = 4 mice per genotype. ( F ) Quantification of interaction between PSD-95 and Mdm2 (* P = 0.0221) after co-immunoprecipitation and representative western blots using lysates from WT primary cortical neuron cultures treated with Aβ42 or Scr (1 µM) for 2 h at DIV 12–14. n = 7 and 6 from two independent cultures for Scr and Aβ42 groups, respectively. ( G ) Representative western blots of Ubiquitin and PSD-95 after immunoprecipitation with anti-PSD-95 antibody using lysates from APP/PS1 mice or their WT littermates at 8 weeks of age. Quantification is performed by first normalizing ubiquitinated PSD-95 (IP: PSD-95, IB: Ub) to immunoprecipitated PSD-95 (IP: PSD-95, IB: PSD-95), followed by normalizing APP/PS1 group to WT group (** P = 0.0081). n = 4 mice per genotype. ( H ) Representative western blots of Ubiquitin and PSD-95 after immunoprecipitation with anti-PSD-95 antibody using lysates from WT primary cortical neuron cultures treated with Aβ42 or Scr (1 µM) for 2 h at DIV 12–14. Quantification is performed by first normalizing ubiquitinated PSD-95 (IP: PSD-95, IB: Ub) to immunoprecipitated PSD-95 (IP: PSD-95, IB:PSD-95), followed by normalizing Aβ42 group to Scr group (*** P = 0.0002). n = 5 from three independent cultures for both groups. Data Information: Significance was determined by Student’s t test ( A – E , G , H ) or Mann–Whitney U test ( F ). Data are represented as mean ± SEM with * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. .

Article Snippet: Mouse anti-PSD-95 antibody (#sc32290), mouse anti-phospho-Mdm2-S166 antibody (#sc-965), and mouse anti-phospho-Mdm2-S186 antibody (#sc-53368) are from Santa Cruz Biotechnology.

Techniques: Western Blot, Immunoprecipitation, Ubiquitin Proteomics, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A ) Representative western blots of PSD-95 and Gapdh from the brain lysates of a group of WT, PSD-95 +/- , APP/PS1, and APP/PS1 × PSD-95 +/− littermate mice. ( B ) Quantification of susceptibility to stage 4 seizures and lethality from 8-weeks-old WT, PSD-95 +/− , APP/PS1, and APP/PS1 × PSD-95 +/− mice intraperitoneally injected with kainic acid (30 mg/kg). For seizure susceptibility, ** P = 0.0016 and * P = 0.0154. For lethality, ** P = 0.0048 and **** P < 0.0001. n = 11–14 mice as indicated. ( C ) Quantification of highest seizure scores (* P = 0.0496) and latency to stage 4 seizures (** P = 0.0031) from 8-weeks-old APP/PS1, and APP/PS1 × PSD-95 +/− mice intraperitoneally injected with kainic acid (30 mg/kg). n = 12 and 14 for APP/PS1, and APP/PS1 × PSD-95 +/− mice, respectively. Sixty minutes in latency to score 4 means the mice without showing score 4 or above. ( D ) Quantification of susceptibility to stage 4 seizures ( P > 0.9999), latency to stage 4 seizures ( P = 0.1552), highest seizure scores ( P > 0.9999), and lethality ( P > 0.9999) from 8-weeks-old WT and PSD-95 +/− mice intraperitoneally injected with kainic acid (45 mg/kg). n = 8 mice for both WT and PSD-95 +/− mice. ( E ) A working model illustrating Mdm2-mediated ubiquitination of PSD-95 reduces seizure susceptibility while impairment of that leads to elevated seizure susceptibility during early Aβ pathology. Data Information: significance was determined by Fisher’s exact test (seizure susceptibility and lethality) or Mann–Whitney U test (highest score and latency to score 4). Data are represented as mean ± SEM with * P < 0.05 and ** P < 0.01, **** P < 0.0001, ns non-significant. .

Article Snippet: Mouse anti-PSD-95 antibody (#sc32290), mouse anti-phospho-Mdm2-S166 antibody (#sc-965), and mouse anti-phospho-Mdm2-S186 antibody (#sc-53368) are from Santa Cruz Biotechnology.

Techniques: Western Blot, Injection, Ubiquitin Proteomics, MANN-WHITNEY