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: Mouse anti-SUMO2/3 , Cytoskeleton, Inc. , Cat# ASM24 RRID: AB_2884969.
Techniques: Staining, Marker, Fractionation, Membrane, Centrifugation, Control, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Western Blot
Proteintech is a verified supplier 
![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 <t>SUMO2.</t> 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5319/pmc12015319/pmc12015319__41419_2025_7524_Fig1_HTML.jpg)
