anti phospho n methyl d aspartate receptors subunit 2b nr2b tyr1472  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: The activity‐dependent protein synthesis is enhanced in DF2‐CKO mice hippocampus. A) Cumulative distribution and violin plot (inside) showing the log 2 ‐fold change in protein levels between DF2‐CKO and control mice in hippocampal tissue under basal conditions. Data are stratified by nontargets versus YTHDF2 RIP targets (Mann–Whitney test, U = 840670, p = 0.770, KS test, Z = 0.741, p = 0.642). B) Cumulative distribution and violin plot (inside) showing the log 2 ‐fold change in protein levels between DF2‐CKO and control mice in hippocampal tissue after fear conditioning. Data are stratified by nontargets versus YTHDF2 RIP targets (Mann–Whitney test, U = 728491, p = 6.78E−6, KS test, Z = 2.495, **** p = 7.87E−6). C) Representative blot (left) and quantification data (right) of puromycin signal in KCl‐treated primary cultured hippocampus neurons from either control or DF2‐CKO mice ( n = 7, 5, 7, 5 wells, one‐way ANOVA, F (3, 20) = 12.64, p <0.0001, post hoc: Turkey's test, Control vs Control + KCl, p = 0.047, Control + KCl vs DF2‐CKO + KCl, p = 0.010). D) Cumulative distribution and violin plot (inside) showing the log 2 ‐translational efficiency (TE) of genes in control and DF2‐CKO mice hippocampus at 1 h after fear conditioning (Mann–Whitney test, U = 2.37E7, p = 2.92E−21, KS test, Z = 6.232, p <0.0001). E) Volcano plot showing the significantly changed TE in DF2‐CKO mice compared to control. Significantly changed TE was defined as log 2 TE > 1 and p < 0.05. F) KEGG and GO enrichment analysis of upregulated proteins identified from hippocampus proteomics at 4 h after fear conditioning. Top ten enriched BP and CC terms were showed. Upregulated proteins were defined as fold change (DF‐CKO/Control) > 1.2 and p <0.05. G,H) Representative western blot (left) and quantification data (right) of GluN2B (G) and SEMA4B (H) expression in PSD fraction and total hippocampal lysates from control and DF2‐CKO mice at 4 h after fear conditioning (G: PSD fraction: n = 4 mice per group, unpaired two‐tailed t ‐test, t 6 = 3.036, p = 0.023; total protein: n = 6 mice per group, unpaired two‐tailed t ‐test, t 10 = 0.329, p = 0.749; H: PSD fraction: n = 4 mice per group, unpaired two‐tailed t ‐test, t 6 = 3.600, p = 0.011; total protein: n = 6 mice per group, unpaired two‐tailed t ‐test, t 10 = 2.492, p = 0.032). Data in violin plots are presented as the median and quartiles. Data in bar plots are presented as mean ± standard error. **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Activity Assay, Control, MANN-WHITNEY, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Enhanced Protein Synthesis and Hippocampus‐Dependent Memory via Inhibition of YTHDF2 ‐Mediated m 6 A mRNA Degradation

doi: 10.1002/advs.202514926

Figure Lengend Snippet: Knockdown of SEMA4B reverses the phenotype of DF2‐CKO mice. A) IGV visualization of YTHDF2 binding peaks on SEMA4B mRNA in YTHDF2 RIP‐seq. The binding peaks were enriched in IP samples compared to input. RIP1 and RIP2 represented two biological replicates of RIP‐seq. B) RIP‐qPCR analysis shows significant enrichment of SEMA4B , Tanc2 , and Grin2b in YTHDF2 ‐immunoprecipitated samples compared to the negative control RPS29 (not identified as a YTHDF2 target in RIP‐seq), indicating specific binding of YTHDF2 to these transcripts. C) The mRNA level of SEMA4B was analyzed by qPCR in Actinomycin‐D (ActD)‐treated primary neurons from control and DF2‐CKO mice ( n = 3 cell dishes per group, repeated two‐way ANOVA, group factor: F (1, 4) = 11.14, p = 0.029, post hoc: Bonferroni's test, 3 h: p = 0.024). D) MeRIP‐qPCR analysis shows significant enrichment of SEMA4B , TANC2 , and Grin2b in m 6 A‐immunoprecipitated samples compared to the negative control Rps21 (not identified as a m 6 A‐modified target in meRIP‐seq), indicating the presence of m 6 A modification on these transcripts. E) Representative blot (left) and quantification data (right) of SEMA4B expression in hippocampus of mice injected with AAV– SEMA4B shRNA ( n = 6, 3, 5, 4 mice, one‐way ANOVA, F (3, 14) = 8.778, p = 0.002, Bonferroni's test: Control + AAV–GFP vs Control + SEMA4B shRNA, p = 0.013, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B shRNA, p = 0.038). F) The freezing curves of DF2‐CKO mice injected with AAV– SEMA4B shRNA during fear conditioning ( n = 20, 11, 10, 10 mice, repeated two‐way ANOVA, group: F (3, 47) = 2.558, p = 0.066). G,H) Contextual (G) and auditory (H) fear memory assessed 1 h after fear training (G: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 7.72, p < 0.001, Bonferroni's test: Control + AAV–GFP vs Control + AAV– SEMA4B ShRNA, p = 0.036, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p = 0.087, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B ShRNA, p = 0.036; H: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 45) = 0.7619, p = 0.521). I,J) Contextual (I) and auditory (J) fear memory assessed 24 h after fear training (I: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 7.838, p < 0.001, Bonferroni's test: Control + AAV–GFP vs Control + AAV– SEMA4B shRNA, p = 0.442, Control + AAV–GFP vs DF2‐CKO + AAV–GFP, p = 0.005, DF2‐CKO + AAV–GFP vs DF2‐CKO + AAV– SEMA4B shRNA, p = 0.030; J: n = 20, 11, 10, 10 mice, one‐way ANOVA, F (3, 47) = 0.1705, p = 0.916). K) Representative traces of sEPSCs. L,M) Cumulative probability plots and bar graph (inside) indicated both decreased sEPSCs frequency (L) and amplitude (M) in CA1 pyramidal neurons from DF2‐CKO mice with AAV– SEMA4B expression ( n DF2‐CKO+AAV–GFP = 26 cells, 3 mice, n DF2‐CKO+AAV–shRNA = 21 cells, 3 mice; unpaired t ‐test, t 45 = 2.652, p = 0.011, KS test, p < 0.0001 for frequency; unpaired t ‐test, t 45 = 4.287, p <0.0001, KS test, p < 0.0001 for amplitude). N) Representative traces of sIPSCs. O,P) Cumulative probability plots and bar graph (inside) showing unaffected inhibitory neurotransmission ( n DF2‐CKO+AAV–GFP = 24 cells, 3 mice, n DF2‐CKO+AAV–shRNA = 19 cells, 3 mice; unpaired t ‐test, t 41 = 0.623, p = 0.537 for frequency; unpaired t ‐test, t 41 = 1.505, p = 0.140 for amplitude). Data are presented as mean ± standard error. * p < 0.001; **** p < 0.0001.

Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature, the transferred membranes were incubated overnight at 4 °C with primary antibodies: anti‐ YTHDF2 (1:1000, Aviva Systems Biology, ARP67917_P050), anti‐ SEMA4B (1:1000, Cell Signaling Technology, 13771S), anti‐DCP1a (1:1000, Proteintech, 22373‐1‐AP‐50), anti‐Tia1 (1:1000, PTM BIO, PTM‐5518), anti‐β‐actin (1:5000, Sigma, A5441), GAPDH (1:5000, Thermo Fisher Scientific, AM4300), GluN2B (1:1000, Cell Signaling Technology, 4207S), Flotillin 1 (Proteintech, 15571‐1‐AP).

Techniques: Knockdown, Binding Assay, Immunoprecipitation, Negative Control, Control, Modification, Expressing, Injection, shRNA