Journal: Cell Death & Disease

Article Title: Opposing roles for GSK3β and ERK1-dependent phosphorylation of huntingtin during neuronal dysfunction and cell death in Huntington’s disease

doi: 10.1038/s41419-025-07524-0

Figure Lengend Snippet: A Representative images from healthy/normal (WT, Q17) or diseased (HD, Q109) human iPSCs stained with the pluripotent marker OCT-4, the neuronal precursor (NPC) marker Nestin and the mature neuronal markers MAP2 and βIII-Tubulin. Hoechst stains nuclei. Scale = 25 μm. Differentiated neurons show Synaptophysin (SYP) positive staining. Scale = 10 μm. B Electrophysiological analysis of WT and HD human neurons differentiated from iPSCs show action potentials, which are abolished in the presence of TTX or TEA. C Schematic diagram of human iNeuron lysate fractionation into perinuclear supernatant (PNS), light membrane (LM), soluble (SF), and heavy membrane (P1) fractions by ultra-centrifugation and sucrose gradient separation. D Workflow for quality control and quantification of unique peptides identified from LC-MS of HTT-IPs from WT or HD human iNeurons. E Hierarchical cluster heat map showing the avg. relative abundance (spectral count; SpC) of 800 proteins (≥3 unique peptides/trial across ≥2 biological replicates) quantified across the WT and HD HTT-IPs with a normalized fold change (FC) threshold of ±2X and a significance threshold of p < 0.05 determined by a Welch’s t test across three independent biological replicates. Increased in HD HTT-IP = red, decreased in HD HTT-IP = blue. In addition, proteins were identified in only WT HTT-IP (lost = green) or in only HD HTT-IP (gained = orange). F Volcano plot with the y axis depicting significance (−log 10 [ p value]) and the x axis depicting fold change of individual peptides between HD and WT HTT-IPs (log 2 [FC]). Three independent biological replicates were performed for each genotype. A negative, no-antibody IP was performed to account for non-specific peptide association with magnetic beads. G Representative western blot of HTT-IP from WT or HD LMs, probed against HTT, KIF5A, KIF5B, KIF5C, DNCT, MAP1B, MAP2, RAB2, RAB5, RAB7, VPS35, or SUMO2. Except for KIF5A, all show presence in WT and HD HTT-IP. No bands are seen in the negative no antibody control (−Crtl). n = 3. Statistical analysis was conducted using the two-sample two-sided Student’s t test comparing signal/noise intensity between bands in WT and HD conditions normalized to WT. Data represented as mean ± SEM. ns = p > 0.05, * p < 0.05, ** p < 0.005.

Article Snippet: Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Thermofisher 1:1000), Rab4 (Abcam 1:1000), Rab5 (Abcam 1:1000), Rab2 (SCBT 1:500), Rab7 (SCBT 1:500), VPS35 (SCBT 1:500), SUMO2/3 (Cytoskeleton 1:500), KIF5A (Goldstein 1:250), KIF5B (Goldstein 1:250), KIF5C (Goldstein 1:250), DIC (Abcam 1:1000), DNCT (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), TOM20 (CellSignaling Technology 1:500), MAP1B (SCBT 1:1000), MAP2 (BD Pharmigen 1:1000), Total AKT1 (CellSignaling Technology 1:1000), pAKT1 (Ser473, CellSignaling Technology 1:1000), Total GSK3α/β (CellSignaling Technology 1:1000), pGSK3α/β (pY279/pY216; Abcam 1:1000), or ERK (pan-ERK; BD Transduction Laboratories 1:1000) for 16 h at 4 °C.

Techniques: Staining, Marker, Fractionation, Membrane, Centrifugation, Control, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Western Blot

Journal: Cell Death & Disease

Article Title: Opposing roles for GSK3β and ERK1-dependent phosphorylation of huntingtin during neuronal dysfunction and cell death in Huntington’s disease

doi: 10.1038/s41419-025-07524-0

Figure Lengend Snippet:

Article Snippet: Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Thermofisher 1:1000), Rab4 (Abcam 1:1000), Rab5 (Abcam 1:1000), Rab2 (SCBT 1:500), Rab7 (SCBT 1:500), VPS35 (SCBT 1:500), SUMO2/3 (Cytoskeleton 1:500), KIF5A (Goldstein 1:250), KIF5B (Goldstein 1:250), KIF5C (Goldstein 1:250), DIC (Abcam 1:1000), DNCT (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), TOM20 (CellSignaling Technology 1:500), MAP1B (SCBT 1:1000), MAP2 (BD Pharmigen 1:1000), Total AKT1 (CellSignaling Technology 1:1000), pAKT1 (Ser473, CellSignaling Technology 1:1000), Total GSK3α/β (CellSignaling Technology 1:1000), pGSK3α/β (pY279/pY216; Abcam 1:1000), or ERK (pan-ERK; BD Transduction Laboratories 1:1000) for 16 h at 4 °C.

Techniques: Transduction, Recombinant, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, In Situ, Software, Imaging

Journal: Nature Communications

Article Title: Structural basis for the human SENP5’s SUMO isoform discrimination

doi: 10.1038/s41467-025-60029-4

Figure Lengend Snippet: a Close-up view of the ribbon representation of interfaces of SENP5 CD -SUMO2 (green) and SENP5 CD -SUMO1 (blue) around Asn607 and Arg624. Interface residues are labelled and shown in stick representation. b Plot of the proteolytic cleavage of SUMO1-AMC and SUMO2-AMC to test the activity of SENP5 CD wild type and SENP5 CD N607A, R624A, K627A, and R624/K627A mutants. SUMO2-AMC was incubated with SENP5 CD , and released AMC was identified by fluorescence. The plot is an average of a triplicate experiment. c SDS-PAGE of the endpoint assays of SENP5 CD wild type and SENP5 CD N607A, R624A K627A, and R624/K627A mutants using RanGAP1-SUMO1 and RanGAP1-SUMO2 substrates. ( Below) Plot of the fraction of the RanGAP1-SUMO2 and RanGAP1-SUMO1 substrate after 30 min reaction. Data values represent the mean ± SEM, n = 3 technical replicates. Significance was measured by a two-tailed unpaired t test relative to wild-type. All data were analyzed with a 95% confidence interval. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Exact P values from left to right: 0.0005, <0.0001, <0.0001, and <0.0001. d Western Blot analysis of HEK293F cells co-transfected with Flag-PML and either the empty pcDNA3 (−) or the plasmids expressing full-length SENP5, SENP5-R624A, SENP5-C713A or SENP5-R624A/K627A. ( left ) Immunoblot showing the levels of endogenous SUMO1 and SUMO2 in HEK293F cells. Anti-SUMO1 and antiSUMO2 antibodies were used to detect both endogenous SUMO isoforms. Anti-tubulin antibody was used as a loading control. ( right ) Plot of the Western Blot triplicate showing the fraction of polySUMO conjugates for each experiment regarding to SENP5 active site mutant (C713A). Data values represent the mean ± SEM, n = 3 technical replicates. Significance was measured by a two-tailed unpaired t test relative to C713A mutant. All data were analyzed with a 95% confidence interval. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Exact P values from left to right: <0.0001 and 0.0032. Source data are provided as a Source Data file.

Article Snippet: Full-length RGS-SENP5 was a gift from Edward Yeh (Addgene plasmid #18053; http://n2t.net/addgene:18053 ; RRID:Addgene_18053), pLPC-Flag-PML-IV was a gift from Gerardo Ferbeyre (Addgene plasmid #62804; http://n2t.net/addgene:62804 ; RRID:Addgene_62804), pcDNA3-HA-SUMO1 was a gift from Junying Yuan (Addgene plasmid #21154; http://n2t.net/addgene:21154 ; RRID:Addgene_21154) and pcDNA3 HA-SUMO2 WT was a gift from Guy Salvesen (Addgene plasmid #48967; http://n2t.net/addgene:48967 ; RRID:Addgene_48967) , – .

Techniques: Activity Assay, Incubation, Fluorescence, SDS Page, Two Tailed Test, Western Blot, Transfection, Expressing, Control, Mutagenesis