Journal: Molecular & Cellular Proteomics : MCP

Article Title: Spinal Cord Phosphoproteome of SCA2 Mouse Model Reveals Alteration of ATXN2-N-Term PRM–SH3–Actin Interactome and of Autophagy

doi: 10.1016/j.mcpro.2025.101072

Figure Lengend Snippet: Quantitative immunoblots ( A , C , E , G , I , N = 5 or 4 versus 5) and quantitative reverse-transcriptase real-time PCR ( B , D , F , H , J , N = 6 versus 6) for SQSTM1 ( A , B ), NBR1 ( C , D ), and OPTN ( E , F ) as main aggrephagy receptors, as well as MYO6 ( G , H ) and SPARCL1 ( I , J ) in end-stage spinal cord from our SCA2–ALS13 mouse model Atxn2 -CAG100-KnockIn (KIN) versus WT. SQSTM1 and NBR1 were chosen in view of their hyperphosphorylated peptides, OPTN in view of its mix of hyperphosphorylations and hypophosphorylations, together with its interactor MYO6, and SPARCL1 in view of its many hypophosphorylated peptides. The protein changes are reflected by similar transcript upregulation for SQSTM1, versus downregulation for SPARCL1, but the MYO6 protein accumulation is counteracted by downregulation of Myo6 mRNA—a possible correlate of deficient autophagosome fusion with lysosomes. ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, ns, not significant. ALS, amotrophic lateral sclerosis; MYO6, unconventional myosin VI; OPTN, optineurin; SCA2, spinocerebellar ataxia type 2; SPARCL1, secreted protein acidic and cysteine rich-like 1.

Article Snippet: The gene expression TaqMan assays (Thermo Fisher Scientific) used for this study were Bnip3 (Mm00833810_g1), Ctsb (Mm01310506_m1), Myo6 (Mm00500651_m1), Nbr1 (Mm01249798_m1), Optn (Mm01333245_m1), Ppia (Mm02342430_g1), Sparcl1 (Mm00447784_m1), Sqstm1 (Mm00448091_m1), Tax1bp1 (Mm00518038_m1), Ubqln2 (Mm00834570_s1), Wdfy3 (Mm00556380_m1), and Wnk1 (Mm01184014_m1, Mm01184007_m1, and Mm01184011_m1).

Techniques: Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Knock-In

Journal: American Journal of Cancer Research

Article Title: USP18 promote tumor immune evasion in pancreatic cancer through enhancing autolysosome-mediated degradation of MHC-I

doi: 10.62347/NDYC1006

Figure Lengend Snippet: USP18 promotes MHC-I trafficking to lysosomes via NBR1. A, B. The interaction of USP18 and HLA-A was analyzed by Co-IP analysis. C. Vector and Flag-USP18 were transfected in PC cells with HA-HLA and treated with MG132 (20 µM) for 4 h. Cellular extracts were immunoprecipitated with anti-HA and followed by Western blot with anti-ubiquitin (Ub) antibody. D. AsPC-1 cells expressing Flag-USP18 and HA-HLA-A were analyzed by Co-IP and Western blot. E. The expression levels of NBR1 and Tubulin were detected by Western blot analysis. F. The expression levels of MHC-I, USp18, NBR1 and Tubulin were detected by Western blot analysis.

Article Snippet: The primary antibodies included anti-USP18 (1:1000, Proteintech, Cat No. 12153-1-AP), anti-NBR1 Monoclonal antibody (1:1000, Proteintech, Cat No. 68062-1-Ig), anti-MHC-I (1:1000, abcam, ab134189), anti-HA (1:1000, Sigma, SAB1306082), anti-Flag (1:1000, Sigma, F1804), anti-GAPDH (1:1000, Abcam, ab8245), and anti-ubiquitin (1:1000, Santa Cruz, sc-53509).

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing

Journal: American Journal of Cancer Research

Article Title: USP18 promote tumor immune evasion in pancreatic cancer through enhancing autolysosome-mediated degradation of MHC-I

doi: 10.62347/NDYC1006

Figure Lengend Snippet: USP18 interacts with NBR1. A. qRT-PCR analysis of USP18 and NBR1 at the mRNA level in USP18 knockdown PC cells. **P < 0.01. B. qRT-PCR analysis of USP18 and NBR1 at the mRNA level in Flag-USP18 PC cells. **P < 0.01. C. The proteins that co-precipitated with USP18 were identified via LC-MS/MS. #PSMs with matching peptide profiles. D, E. The interaction of USP18 and HLA-A was analyzed by Co-IP analysis.

Article Snippet: The primary antibodies included anti-USP18 (1:1000, Proteintech, Cat No. 12153-1-AP), anti-NBR1 Monoclonal antibody (1:1000, Proteintech, Cat No. 68062-1-Ig), anti-MHC-I (1:1000, abcam, ab134189), anti-HA (1:1000, Sigma, SAB1306082), anti-Flag (1:1000, Sigma, F1804), anti-GAPDH (1:1000, Abcam, ab8245), and anti-ubiquitin (1:1000, Santa Cruz, sc-53509).

Techniques: Quantitative RT-PCR, Knockdown, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay

Journal: American Journal of Cancer Research

Article Title: USP18 promote tumor immune evasion in pancreatic cancer through enhancing autolysosome-mediated degradation of MHC-I

doi: 10.62347/NDYC1006

Figure Lengend Snippet: USP18 stabilizes NBR1 protein expression via suppressing NBR1 degradation mediated by proteasome. A. NBR1 protein levels at various times were measured by western blotting after MG132 addition (10 µM) to AsPC-1 and PANC-1 cells. B, C. Western blot analysis of USP18 and NBR1 protein expression in PANC-1 cells transfected with shUSP18 or shNC and AsPC-1 cells transfected with exogenous USP18 or a control vector, with or without treatment with 10 µM MG132. D-G. PANC-1 cells transfected with shUSP18 or shNC and AsPC-1 cells transfected with exogenous USP18 or a control vector were subjected to treatment with 20 µg/mL CHX, followed by assessment of NBR1 protein levels using western blotting. **P < 0.01. H. Lysates from PC cells transduced with shUSP18 or Flag-USP18 were immunoprecipitated with the anti-Ub and immunoblotted with the anti-NBR1. Cells were treated with MG132 for 6 h before collection.

Article Snippet: The primary antibodies included anti-USP18 (1:1000, Proteintech, Cat No. 12153-1-AP), anti-NBR1 Monoclonal antibody (1:1000, Proteintech, Cat No. 68062-1-Ig), anti-MHC-I (1:1000, abcam, ab134189), anti-HA (1:1000, Sigma, SAB1306082), anti-Flag (1:1000, Sigma, F1804), anti-GAPDH (1:1000, Abcam, ab8245), and anti-ubiquitin (1:1000, Santa Cruz, sc-53509).

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Transduction, Immunoprecipitation