human cd63 cd9 alix antibody  (Proteintech)

Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: A potential multimodal biomarker – cognitive signature associated with the conversion from subjective cognitive decline to mild cognitive impairment

doi: 10.1002/trc2.70240

Figure Lengend Snippet: Characterization and imaging of serum‐derived cEVs. (A) cEVs images as acquired by transmission electron microscopy; bar represents 100 nm. (B) Concentration and size distribution of cEVs examined by NTA using Nanosight‐NS300. (C) ApoA1 concentration in serum and cEVs. (D) Immunoblot detection of EV protein markers and non‐associated proteins. The parametric Student's t ‐test was used for statistical analysis of ApoA1; *** p < 0.001. ALIX, ALG‐2‐interacting protein X; ApoA1, apolipoprotein A1; CD63, cluster of differentiation 63; cEVs, circulating extracellular vesicles; CTR, controls; nm, nanometer; NTA, nanoparticle tracking analysis; SCD, subjective cognitive decline; TSG101, tumor susceptibility gene 101.

Article Snippet: Proteins were incubated for 30 min with primary antibodies against TSG101 (Novus Biologicals LLC, Catalog No.: NB200–112, 1:10), CD63 (R&D Systems; Catalog No.: MAB50482, 1:20), ALIX (R&D Systems; Catalog No.: MAB50482, 1:20), and calnexin (Novus Biologicals LLC; Catalog No.: NB100–1965, 1:25), followed by washing and incubation with species‐specific secondary antibodies.

Techniques: Imaging, Derivative Assay, Transmission Assay, Electron Microscopy, Concentration Assay, Western Blot

Journal: Advanced Science

Article Title: Tumor‐Derived CDC37 Inhibits Antigen Cross‐Presentation in Dendritic Cells and Impairs Anti‐Tumor Immunity in Breast Cancer

doi: 10.1002/advs.202506518

Figure Lengend Snippet: Tumor EVs shuttled CDC37 to DC endosomes to suppress antigen cross‐presentation. A,B) DCs were treated with EVs from TMB hi CTL hi or TMB hi CTL lo breat tumors, with or without the ERAD inhibitor Eeyarestatin I (EeyI). A) Representative flow cytometric plots and quantification of AnnexinV + PI − DCs after exogenous cytC treatment. B) Representative flow cytometric plots and quantification of GZMB + CD8 + T cells primed by DCs with indicated treatment. C,D) DCs were treated with PBS or EVs from TMB hi CTL lo ( n = 3) and TMB hi CTL hi ( n = 3) tumors, respectively. C) Heatmap displaying differentially expressed proteins in the endosomes of DCs with indicated treatment, determined by targeted mass spectrometry focusing on 18 proteins of interest, potentially related to ERAD in endosomes. Color scale, log 2 (fold change) normalized to endosome marker EEA1. D) Representative images of CDC37 expression in the endosomes and whole cell of DCs with indicated treatment, are shown by western blotting ( n = 3 independent experiments). Na/K ATPase was used as a marker of cell membrane protein and EEA1 was used as a marker of endosomes. MW, molecular weight. E) Representative images of immunofluorescence staining for CDC37 and RAB5 in TiDCs of TMB hi CTL lo ( n = 3) and TMB hi CTL hi ( n = 3) breast tumor patients. Scale bar, 5 µm. Quantification is shown in Figure (Supporting Information). F–I). DCs were transduced with control sgRNA (sgNC), sgRNAs targeting CDC37 (CDC37‐sg1, CDC37‐sg2) and then treated with EVs from TMB hi CTL lo ( n = 3) or TMB hi CTL hi ( n = 3) breast tumor, respectively. F) Representative immunofluorescence images of CDC37 and RAB5 co‐staining in DCs. Scale bar, 5 µm. Quantification is shown in Figure (Supporting Information). G) Cell apoptosis induced by endosome‐cytosol export of internalized cytC in DCs were determined by flow cytometry. Percentages of AnnexinV + PI − DCs are shown. H) Quantification of β‐lactamase activity represented by ratiometric values (450 nm: 535 nm) of CCF4 in DCs, determined by flow cytometry. I) DCs with indicated treatment were pulsed with tumor lysates and co‐cultured with autologous naive CD8 + T cells, respectively. Representative flow cytometric plots and quantification of the percentages of GZMB releasing CD8 + T cells. J) Representative images of CDC37 expression in tumor tissue, or in the supernatant and EV components of TCM from TMB hi CTL lo ( n = 3) and TMB hi CTL hi ( n = 3) breast cancer patients, are shown by western blotting ( n = 3 independent experiments). ALIX and CD81 represent the markers of EVs, and ponceau S represents total protein loading for normalization. K) Representative live imaging for GFP‐RAB5 overexpressing DCs treated with EVs of mCherry‐CDC37 overexpressing SKBR3 cancer cells during 21 min ( n = 3 independent experiments). Scale bar, 5 µm. Quantification of co‐localization of CDC37 and RAB5 at indicated time point is shown. Results are mean ± s.d. of independent experiments producing similar results. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with TMB hi CTL lo group (A,B), DCs transduced with indicated sgRNA and treated with PBS (G‐I), or DCs without EV treatment (0 min) (K), were determined by two‐tailed Student's t test (A, B), or two‐tailed one‐way ANOVA with Dunnett's multiple‐comparisons test (G–I,K).

Article Snippet: Membranes were blocked in 5% BSA for 30 min at room temperature and then incubated at 4 °C with the following primary antibodies: rabbit anti‐human HSP90 (Cat# 4877, CST, 1:1000), mouse anti‐human CDC37 (Cat# 66 420, Proteintech, 1:1000), mouse anti‐human MUC‐1 (Cat# 4532, Immunoway, 1:1000), rabbit anti‐human Alix (Cat# 12422‐1‐AP, Proteintech, 1:1000), mouse anti‐human CD81 (Cat# 66866‐1‐Ig, Proteintech, 1:1000), rabbit anti‐human CD63 (Cat# 5702, Abcam, 1:1000), rabbit anti‐human EEA1 (Cat# 4245, Abcam, 1:1000), rabbit anti‐human Na/K ATPase (Cat# 1845Y, Abcam, 1:1000), rabbit anti‐human PSME2 (Cat#ab183727, Abcam, 1:1000), mouse anti‐human CHIP (Cat# sc‐133066, Santa Cruz, 1:500), mouse anti‐human GAPDH (Cat# 60 004, Proteintech, 1:10 000).

Techniques: Mass Spectrometry, Marker, Expressing, Western Blot, Membrane, Molecular Weight, Immunofluorescence, Staining, Transduction, Control, Flow Cytometry, Activity Assay, Cell Culture, Imaging, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Dynamic Change of PD‐L2 on Circulating Plasma Extracellular Vesicles as a Predictor of Treatment Response in Melanoma Patients Receiving Anti‐PD‐1 Therapy

doi: 10.1002/jev2.70054

Figure Lengend Snippet: The workflow and extracellular vehicle (EV) characteristics. (a) A schematic representation of the entire study is shown. (b) Schematic (top) for EV membrane protein array to investigate the 45 key proteins in the discovery cohort and schematic (bottom) for ELISA to measure the target protein concentration on the surface of EVs isolated from the validation cohort. (c) A representative TEM image illustrating EVs derived from the plasma of melanoma patients. Scale bar, 200 nm. (d) Concentration and size distribution of purified plasma EVs using NanoFCM. (e) A representative western blot image for EVs markers. CD9, CD81 and Alix were used as specific EV markers, and Calnexin as a negative marker. All lanes were loaded with the same amount of total protein.

Article Snippet: Antibodies include anti‐mouse CD9 (31980, Thermo Fisher, USA), anti‐mouse CD81 (79004, Thermo Fisher, USA), anti‐human/mouse Alix (sc‐53540, Santa Cruz, USA), anti‐human CD9 (ab236630, Abcam, USA), anti‐human CD81 (ab79559, Abcam, USA), anti‐human/mouse Calnexin (sc‐46669, Santa Cruz, USA), HRP Goat Anti Rabbit IgG (Immunoway, RS0002) and HRP Goat Anti Mouse IgG (Immunoway, RS0001).

Techniques: Membrane, Protein Array, Enzyme-linked Immunosorbent Assay, Protein Concentration, Isolation, Biomarker Discovery, Derivative Assay, Clinical Proteomics, Concentration Assay, Purification, Western Blot, Marker