cycloheximide chx chase assay  (TargetMol)

Journal: Scientific Reports

Article Title: Dlgap2 deficiency disrupts synaptic homeostasis by promoting ubiquitin-mediated Itsn1 degradation in a valproic acid-induced autism-like model

doi: 10.1038/s41598-026-39099-x

Figure Lengend Snippet: Knockdown of Dlgap2 triggers ubiquitin-mediated degradation of Itsn1. (A) An interaction between Dlgap2 and Itsn1 was detected by co-immunoprecipitation using anti-Dlgap2 antibody on whole-brain synaptoneurosomal lysates. n = 2 samples per group. (B-C) Knockdown of Dlgap2 leads to a decrease in Itsn1 levels within the postsynaptic compartment (PSD fraction) from DIV 9 primary cortical neurons. n = 6 samples per group. (D-E) Knockdown of Dlgap2 reduces protein levels of Itsn1, PSD95, and Homer1 in synaptoneurosomes from shDG2 -injected mice compared with shCon controls. n = 4 samples per group. (F-G) Dlgap2 downregulation promotes the K48-mediated ubiquitin-proteasome-mediated degradation of Itsn1 in primary cortical neurons (DIV 9). n = 4 samples per group. (H-I) Treatment with MG132 restored Itsn1 expression in CHX-treated Dlgap2 knockdown cortical neurons. n = 4 samples per group. (Data represent mean ± SEM. ns: not significant. p < 0.05 indicates significance between the two indicated groups. C , E , G : Two-way ANOVA with post hoc Tukey’s test. I : One-way ANOVA with post hoc Tukey’s test)

Article Snippet: Protein degradation was assessed using a CHX chase assay (Cell Signaling Technology, #2112). shDlgap2 interfering primary cortical neurons were treated with cycloheximide (150 ng/μl) and MG132 (10 μM) to block de novo protein synthesis and inhibited the ubiquitination-mediated degradation.

Techniques: Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Injection, Expressing