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. 2024 Apr 1;300(5):107253. doi: 10.1016/j.jbc.2024.107253

Figure 2.

Figure 2

Homocysteine mediated increase in Pyk2 and SFK phosphorylation leads to GluN2A-NMDAR phosphorylation in neurons.A–F, rat neuron cultures were treated with 50 μM L-homocysteine (L-Hcy, 4 h) in the presence and absence of Pyk2 inhibitor PF431396 (5 μM) or SFK inhibitor PP2 (5 μM). A, GluN2A-subunit of NMDAR was immunoprecipitated from total cell lysate using anti-GluN2A antibody, and the immune complexes were processed for immunoblot analysis using anti-pGluN2AY1325 antibody (upper panel). Total cell lysates were immunoblotted with anti-β-tubulin antibody to ensure equal input protein for immunoprecipitation (lower panel). One-way ANOVA revealed significant main effect of treatment (F (3, 8) = 146.3; p < 0.0001). B–F, total cell lysates were processed for immunoblot analysis using (B and D) anti-phospho-Pyk2 Y402 (pPyk2) and anti-Pyk2 antibodies or (C, E, and F) anti-phospho-Src Y416 (pSrc) and anti-Src antibodies. B, C, and F, one-way ANOVA revealed significant main effect of treatment in the presence of (B) Pyk2 inhibitor: F (2, 6) = 50.27; p = 0.0002, (C) SFK inhibitor: F (2, 6) = 59.18; p = 0.0001, and (F) Pyk2 inhibitor: F (2, 6) = 34.94; p = 0.0005. G and H, rat neuron cultures were treated with 50 μM L-Hcy (4 h) in the presence and absence of GluN2A-NMDAR inhibitor NVP-AAM077 (30 nM) or GluN2B-NMDAR inhibitor Ro25-6981 (5 μM). Total cell lysates were processed for immunoblot analysis using (G) anti-pPyk2 and anti-Pyk2 antibodies or (H) anti-pSrc and anti-Src antibodies. I–L, rat neuron cultures were treated with either (I and J) 50 μM L-Glutamate (0 min, 5 min, and 30 min) or (K and L) 50 μM L-Hcy (0 min, 5 min, 0.5 h, 1 h, 2 h, and 4 h). Total cell lysates were processed for immunoblot analysis using (I and K) anti-STEP and anti-tubulin antibodies or (J and L) anti-pSrc and anti-Src antibodies. A, B, C, and E, post hoc analysis by Bonferroni’s multiple-comparisons test shows ∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.001 between the treatment groups. Values are mean ± SD. Data points represent individual biological replicates. NMDAR, N-methyl-D-aspartate subtype of glutamate receptor; SFK, Src family kinase.