Journal: RNA Biology

Article Title: Impact of Gemin5 in protein synthesis: phosphoresidues of the dimerization domain regulate ribosome binding

doi: 10.1080/15476286.2025.2540654

Figure Lengend Snippet: Substitutions of Ser or Thr residues decrease protein stability. (A) Diagram of the Gemin5 protein showing its main functional domains, with residue numbers marking the boundaries of each domain. A detailed alignment of the amino acid sequence (residues 800–875) across selected species is provided. Positions highlighted in red indicate phosphorylation sites identified in the PhosphoSite database. (B, C) HEK293 cells expressing the wild-type version of Gemin5 (G5) (B), side by side with mutants S847A, S847D, STS/AAA and STS/DDD for 24 h (12 h in the case of G5 845–1508 , G5C) (C) were treated with cycloheximide (CHX) for additional 16 h. Samples were taken at 0, 12, and 16 h post CHX treatment. The intensity of each protein at the indicated time was determined by WB. Bars represent the protein intensity (mean ± SEM) of three independent experiments relative to time 0 in each case. Asterisks denote P-values (* p < 0.05, * *p < 0.01, ** *p < 0.001).

Article Snippet: For cycloheximide (CHX) chase experiments, CHX (100 μg/ml) (Merck) was added to halt translation at 12 or 24 h post-transfection, for Xpress-G5C or Xpress-G5 proteins, respectively.

Techniques: Functional Assay, Residue, Sequencing, Phospho-proteomics, Expressing

Journal: Frontiers in Immunology

Article Title: Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

doi: 10.3389/fimmu.2016.00514

Figure Lengend Snippet: Similar mRNA expression levels, protein levels, and protein half-life of SOCS1 and SOCS1ΔNLS. (A) Total Socs1 mRNA expression levels in BMMs of Socs1 +/− , Socs1 +/− MGL tg , and Socs1 −/− MGL tg mice upon stimulation with IFNγ (50 ng/ml) for 24 h normalized to Actin (ActB) expression ( n = 4, mean + SD, two-way ANOVA including Bonferroni post-test). (B) Detection of SOCS1 protein in Socs1 −/− , Socs1 +/− , and Socs1 −/− MGL tg mice upon stimulation with IFNγ (50 ng/ml) for 6 h using anti-SOCS1 hybridoma cell supernatant. Arrows indicate band for SOCS1 and SOCS1ΔNLS. (C) Quantification of SOCS1 protein for Socs1 +/− mice and of SOCS1ΔNLS protein for Socs1 −/− MGL tg mice compared to β-actin was done using ImageJ ( n = 10–13, mean + SD, Student’s t -test). (D) Protein stability was assessed performing a cycloheximide (CHX) chase. CHX (100 μg/ml) was added at 6 h poststimulation with IFNγ and BMMs were lysed at the indicated timepoints and analyzed for SOCS1 expression by Western Blotting. (E) SOCS1 and SOCS1ΔNLS protein expression was normalized to β-actin in order to calculate the percentage of remaining protein relative to its expression prior to the addition of CHX. IkBα was used as a control for effective CHX treatment ( n = 3–5, mean + SD, two-way ANOVA including Bonferroni post-test).

Article Snippet: For cycloheximide (CHX) chase, 1 × 10 6 BMMs were stimulated with IFNγ for 6 h and chased with 100 μg/ml CHX (Merck Millipore, MA, USA).

Techniques: Expressing, Western Blot, Control

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