Journal: Neoplasia (New York, N.Y.)

Article Title: Oral bioavailable ITRI-148 degrades androgen receptor variants and overcomes antiandrogen resistance in advanced prostate cancer

doi: 10.1016/j.neo.2025.101253

Figure Lengend Snippet: ITRI-148 induces degradation of both AR and AR-V7 proteins in CRPC cells. (A) Chemical structures of ITRI-148 and its inactive analog. (B) Expression of Flag-tagged AR-V7 in 293T cells following ITRI-148 treatment. Cells transiently expressing Flag-AR-V7 were pretreated with 50 μg/mL cycloheximide for 1 hour, then exposed to ITRI-148 at the indicated concentrations for 8 hours. AR-V7 expression was detected using an anti-Flag antibody. (C) Representative immunoblots showing AR protein levels in VCaP and CWR22Rv1 cells after 48 hours of ITRI-148 treatment. An N-terminal AR antibody was used to detect both full-length AR and LBD-truncated variants (AR-V(ΔLBD)) in CWR22Rv1 cells, while AR-V7 was detected using an AR-V7-specific antibody. DC₅₀ values were calculated from normalized band intensities, and Dₘₐₓ represents the maximal degradation achieved at 10 μM ITRI-148. (D) Immunofluorescence staining of AR after 24 or 48 hours of ITRI-148 treatment, detected using an N-terminal AR antibody. (E) Western blot analysis of AR and AR-V7 expression in cells treated with DMSO (mock), 10 μM ITRI-148, or the inactive analog. (F) Western blot analysis of ectopically expressed wild-type (WT) and mutant Myc-tagged AR in DU145 cells following 24 hours of treatment with 10 μM ITRI-148. Quantified AR levels are as indicated.

Article Snippet: CWR22Rv1, VCaP, DU145, HEK293T, LNCaP, and PC3 were obtained from ATCC; PNT2 from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: Oral bioavailable ITRI-148 degrades androgen receptor variants and overcomes antiandrogen resistance in advanced prostate cancer

doi: 10.1016/j.neo.2025.101253

Figure Lengend Snippet: ITRI-148 induces AR degradation through a ubiquitin-proteasome-dependent mechanism. (A) Proximity ligation assay (PLA) detecting the physical interaction between AR and CRBN upon ITRI-148 treatment. 293T cells transiently expressing Flag-AR or Flag-AR∆LBD were treated with DMSO or ITRI-148 for 8 hours, followed by PLA detection using anti-Flag and anti-CRBN antibodies. (B) Ubiquitination of AR and AR∆LBD proteins. 293T cells transfected with Flag-AR or Flag-AR∆LBD and HA-Ub were incubated with ITRI-148 and MG132. AR proteins were immunoprecipitated using anti-Flag, and ubiquitination of the AR proteins was detected by western blotting with anti-HA. (C) Western blot of Flag-AR and Flag-AR∆LBD in DU145 cells treated with ITRI-148 alone or in combination with MG132 (5 μM), MLN4924 (0.5 mM), or lenalidomide (80 μM). Inhibitors were added for 8 or 10 hours after the 24-hour ITRI-148 treatment. AR expression was detected using anti-Flag antibody.

Article Snippet: CWR22Rv1, VCaP, DU145, HEK293T, LNCaP, and PC3 were obtained from ATCC; PNT2 from Sigma-Aldrich.

Techniques: Ubiquitin Proteomics, Proximity Ligation Assay, Expressing, Transfection, Incubation, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Enhancing proteasome activity by NMDAR antagonists explains their therapeutic effect in neurodegenerative and mental diseases

doi: 10.1038/s41598-024-84479-w

Figure Lengend Snippet: Modulation of intrinsically disordered proteins (IDPs) Levels and Proteasome Activity by NMDA Receptor Antagonists.( A ) Ketamine (500 µM) and ( B ) memantine (10 µM) reduce the levels of the cell cycle IDPs p21, p53, and p27 in HepG2 cells. ( C , D ) Western blot analysis confirms that ketamine induces the downregulation of Alzheimer’s disease-associated intrinsically disordered proteins (IDPs), including phosphorylated Tau (pTau) in HepG2 and SH-SY5Y cells and β-amyloid precursor protein ectopically expressed in Hep3B and SH-SY5Y cells. ( E ) The "Intracellular Fluorogenic Proteasome Test" indicates an increase in chymotrypsin-like, caspase-like, and trypsin-like proteasome activities upon treatment with NMDA receptor antagonists. ( F ) Chymotrypsin-like, caspase-like, and trypsin-like proteasome activities measured in whole cells using the "Cell-Based Proteasome Luciferase System" from Promega. ( G ) Assessment of proteasome activity in HepG2 cells treated with additional NMDA receptor antagonists (memantine, MK801-20 µM), a proteasome activator (IU1-50 µM), and an inhibitor (MG132-0.5 µM). ( H – J ) Proteasome inhibition by MG132 prevents the reduction of IDPs such as p21, p53, and pTau caused by NMDA receptor antagonists. ( K , L ) Protein changes in the synapse following single dose of ketamine administration (30 mg/kg) were evaluated by comparing two groups, each composed of three mice. To identify the protein alterations attributed to ketamine, a comparative analysis was conducted against a control group. These alterations were then validated using the SynGO database in conjunction with the dataset provided by van Oostrum et al. (excel Table S1). ( K ) Venn diagram analysis, conducted using the FunRich program, was used to visualize the data. ( L ) Subsequently, enrichment analysis via Enrichr-SynGO of the altered proteome identified significant clustering of these proteins across over 100 synaptic structures. Relevant data are provided in excel Table S2. (M) Venn diagram analysis, conducted using the FunRich program, was used to visualize the data from Excel Table S4 related to intrinsically disordered proteins (IDPs) found in the decreased and increased proteins of the synaptic structures. All proteasome activity tests are repeated with at least two independent experiments, and each experiment contains four replicates. *Asterisks denote significance levels: *p < 0.05, **p < 0.01, ***p < 0.001. P values were calculated using a two-tailed unpaired t-test to compare the control group with the individual chemical effect. Western blot analyses were performed on independent biological samples with the entire procedure replicated at least twice to confirm consistency of the results. All unprocessed and uncropped gel and blot images are available in the supplementary file titled “Unprocessed Gel Blots”.

Article Snippet: Various chemicals and kits were utilized: MG132, RA190, Gliotoxin, Capzimin, (+ /-)-1-(1,2-Diphenylethyl) piperidine maleate, TCS 46b, Eliprodil, AP4, N20C hydrochloride, O-Phospho-L-serine, L-701,324, D-Aspartic acid, Kynurenic acid, UBP 301, (R)-3,4-DCPG, gammaDGG, DL-Glutamic acid monohydrate, L-Glutamic acid monosodium salt (Santa Cruz); Lactacystine, Ketamin hydrochloride, Suc-LLVY-AMC, P005091, Vialin A, Spautin A, ML 323, SKPIN C1, SZL-P1-411, SMER3, SKPIN C1, NCC697923, PYR41, IU1, Lactacystin (Cayman Chemical); Memantine hydrochloride, dizocilpine maleate (MK-801), Wortmannin, MK2206, cycloheximide (Sigma-Aldrich); Z-LLE-AMC, Ac-RLR-AMC (AdipoGen Life Sciences); b-AP15 (MedChemExpress), and Proteasome luciferase system (Promega); ( +)-MK 801, Felbamate, Gavestinel, N-Methyl-D-Aspartic acid, ACPT-II (Tocris Bioscience); Leupeptin, N-Ethylmaleimide, rProteinA/G beads, Ni–NTA Ultra-Flux Affinity Microsphere (Solarbio), and 20S Proteasome Assay Kit (Enzo Life Sciences).

Techniques: Activity Assay, Western Blot, Luciferase, Inhibition, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Enhancing proteasome activity by NMDAR antagonists explains their therapeutic effect in neurodegenerative and mental diseases

doi: 10.1038/s41598-024-84479-w

Figure Lengend Snippet: Antagonist and Agonist Chemicals Associated with Glutamate Receptors, Their Binding Sites, and Effects on Proteasome Activity and p21 Protein Levels.

Article Snippet: Various chemicals and kits were utilized: MG132, RA190, Gliotoxin, Capzimin, (+ /-)-1-(1,2-Diphenylethyl) piperidine maleate, TCS 46b, Eliprodil, AP4, N20C hydrochloride, O-Phospho-L-serine, L-701,324, D-Aspartic acid, Kynurenic acid, UBP 301, (R)-3,4-DCPG, gammaDGG, DL-Glutamic acid monohydrate, L-Glutamic acid monosodium salt (Santa Cruz); Lactacystine, Ketamin hydrochloride, Suc-LLVY-AMC, P005091, Vialin A, Spautin A, ML 323, SKPIN C1, SZL-P1-411, SMER3, SKPIN C1, NCC697923, PYR41, IU1, Lactacystin (Cayman Chemical); Memantine hydrochloride, dizocilpine maleate (MK-801), Wortmannin, MK2206, cycloheximide (Sigma-Aldrich); Z-LLE-AMC, Ac-RLR-AMC (AdipoGen Life Sciences); b-AP15 (MedChemExpress), and Proteasome luciferase system (Promega); ( +)-MK 801, Felbamate, Gavestinel, N-Methyl-D-Aspartic acid, ACPT-II (Tocris Bioscience); Leupeptin, N-Ethylmaleimide, rProteinA/G beads, Ni–NTA Ultra-Flux Affinity Microsphere (Solarbio), and 20S Proteasome Assay Kit (Enzo Life Sciences).

Techniques: Binding Assay, Activity Assay

Journal: Scientific Reports

Article Title: Enhancing proteasome activity by NMDAR antagonists explains their therapeutic effect in neurodegenerative and mental diseases

doi: 10.1038/s41598-024-84479-w

Figure Lengend Snippet: NMDA Receptor Antagonists Do Not Influence Ubiquitination within the Ubiquitin–Proteasome System (UPS), ( A ) Efficacy of various ubiquitination-related molecules (refer to Supplementary Table S2) on Ub-YFP degradation in Hep3B cells. ( B ) Impact of PYR-41 (10 µM-12 h incubation), ketamine (500 µM), and their combination on Ub-YFP degradation. ( C and D ) Modulation of endogenous p21 degradation by PYR-41, ketamine, and memantine in HepG2 cells, both separately and in combination. ( E ) Differential effects of PYR-41 and ketamine on GFP levels in Hep3B cells with p21-GFP, observed via fluorescence microscopy with Dox induction. ( F ) Alterations in GFP and p21 levels by ketamine in the presence of PYR-41 in p21-GFP-expressing Hep3B cells, shown by Western blot. ( G ) Influence of DM-CUL1 on p53 versus p21 and p27 highlighting SCF E3 ligase’s role in ubiquitination, and how ketamine affects p53 degradation in this context. All unprocessed and uncropped gel and blot images are available in the supplementary file titled “Unprocessed Gel Blots”.

Article Snippet: Various chemicals and kits were utilized: MG132, RA190, Gliotoxin, Capzimin, (+ /-)-1-(1,2-Diphenylethyl) piperidine maleate, TCS 46b, Eliprodil, AP4, N20C hydrochloride, O-Phospho-L-serine, L-701,324, D-Aspartic acid, Kynurenic acid, UBP 301, (R)-3,4-DCPG, gammaDGG, DL-Glutamic acid monohydrate, L-Glutamic acid monosodium salt (Santa Cruz); Lactacystine, Ketamin hydrochloride, Suc-LLVY-AMC, P005091, Vialin A, Spautin A, ML 323, SKPIN C1, SZL-P1-411, SMER3, SKPIN C1, NCC697923, PYR41, IU1, Lactacystin (Cayman Chemical); Memantine hydrochloride, dizocilpine maleate (MK-801), Wortmannin, MK2206, cycloheximide (Sigma-Aldrich); Z-LLE-AMC, Ac-RLR-AMC (AdipoGen Life Sciences); b-AP15 (MedChemExpress), and Proteasome luciferase system (Promega); ( +)-MK 801, Felbamate, Gavestinel, N-Methyl-D-Aspartic acid, ACPT-II (Tocris Bioscience); Leupeptin, N-Ethylmaleimide, rProteinA/G beads, Ni–NTA Ultra-Flux Affinity Microsphere (Solarbio), and 20S Proteasome Assay Kit (Enzo Life Sciences).

Techniques: Ubiquitin Proteomics, Incubation, Fluorescence, Microscopy, Expressing, Western Blot

Journal: Cell reports

Article Title: APP-Mediated Signaling Prevents Memory Decline in Alzheimer’s Disease Mouse Model

doi: 10.1016/j.celrep.2019.03.087

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-APP-CTM1 Dr. Gopal Thinakaran ( Vetrivel et al., 2009 ) N/A Rabbit polyclonal anti-APP-CT11 Dr. Gopal Thinakaran ( Vetrivel et al., 2009 ) N/A Rabbit polyclonal anti-APP-NT452 Dr. Gopal Thinakaran ( Vetrivel et al., 2009 ) N/A Mouse monoclonal anti-N-terminal Aβ antibody (clone 3D6) Dr. Dale Schenk (Elan Corporation PLC2) ( Andrew et al. 2017 ) N/A Mouse monoclonal anti-flag (clone M2) Sigma Aldrich Cat#F3165; RRID:AB_259529 Rabbit polyclonal MAP2 Sigma-Aldrich Cat#M3696; RRID:AB_1840999 Mouse monoclonal GAPDH Abeam Cat#ab8245; RRID:AB_2107448 Mouse monoclonal anti-HA (clone 6E2) Cell Signaling Cat#2367; RRID:AB_10691311 Goat anti-mouse Alexa 488 Invitrogen Cat#A-11029; RRID:AB_2534088 Goat anti-mouse Alexa 555 Invitrogen Cat#A-21424; RRID:AB_141780 Goat anti-rabbit Alexa 555 Invitrogen Cat#A-21429; RRID:AB_141761 IRDye 680LT donkey anti-mouse LI-COR Biosciences Cat# 926–68022; RRID:AB_10715072 IRDye 680 donkey anti-rabbit LI-COR Biosciences Cat# 926–32223; RRID:AB_621845 IRDye 800CW donkey anti-mouse LI-COR Biosciences Cat# 926–32212; RRID:AB_621847 IRDye 800CW goat anti-rabbit LI-COR Biosciences Cat#827–08365; RRID:AB_01796098 Aβ end-specific sandwich ELISA using end-specific antibodies (2.1.3: Aβ 42; 13.1.1: Aβ 40 for capture) and Ab5-HRP (pan Aβ) for detection Dr. Todd Golde ( Chakrabarty et al., 2015 ) N/A Mouse monoclonal Aβ anti-B608 for capture and anti-B436 for detection of sandwich ELISA Dr. Steven Wagner ( Vetrivel et al., 2009 ) N/A Bacterial and Virus Strains rAAV2/8 caps id packaging Dr. Todd Golde ( Chakrabarty et al., 2013 ) N/A Chemicals, Peptides, and Recombinant Proteins γ-secretase inhibitor Compound E Dr. Todd Golde Mayo Clinic core facility ( Seiffert et al., 2000 ) N/A Cis-N-(2-phenylcyclopentyl) azacyclotridec-1-en-2-amine (MDL-12,330A) Enzo Life Science Cat# BML-CN 145–0005 cOmplete EDTA-free - EDTA-free protease inhibitor cocktail Roche/Sigma-Aldrich Cat# 04693132001 PhosSTOP EasyPack - phosphatase inhibitor mixture Roche/Sigma-Aldrich Cat# 4906845001 Vectashield mounting medium Vector Laboratories Cat#H-1000 Lipofectamine 2000 Invitrogen/ThermoFisher Cat#11668019 LipoD293TM SignaGen Laboratories Cat# SL100668 Streptavidin-agarose beads Pierce/ThermoFisher Cat#20347 G-agarose beads Pierce/ThermoFisher Cat#20398 Premium sulfo NHS-SS-biotin Pierce/ThermoFisher Cat#PG82077 Experimental Models: Cell Lines HeLa cells expressing APP-FL containing the Swedish mutation (APPSwe) Dr.

Techniques: Sandwich ELISA, Virus, Recombinant, Protease Inhibitor, Plasmid Preparation, Expressing, Mutagenesis, Transgenic Assay, Knock-Out, Software