Journal: International Journal of Molecular Sciences

Article Title: Proteasome-Enriched hPPSCs-Derived EVs Attenuate Hypoxic Injury in Endothelial Cells via Proteasome-Mediated HIF-1α Degradation

doi: 10.3390/ijms27157013

Figure Lengend Snippet: Proteasome composition detection and hPPSCs-EVs activity. ( A ) Protein expression levels of 20S proteasome, PSMD11, and PSMB5. ( B ) Quantitative analysis of 20S proteasome, PSMD11, and PSMB5 protein. ( C ) Proteasome chymotrypsin-like activity assay. n = 3, p < 0.05.

Article Snippet: The antibodies employed for Western blot in this study are listed below: Anti-ALIX antibody (Abcam, ab275377, rabbit, Cambridge, MA, USA), diluted at 1:1000; Anti-CD81 antibody (ABclonal, A5270, rabbit, Woburn, MA, USA), diluted at 1:1000; Anti-CD63 antibody (ABclonal, A19023, rabbit), diluted at 1:1000; Anti-HIF-1α antibody (ABclonal, A16873, rabbit), diluted at 1:1000; Anti-Ubiquitin (P4D1) antibody (SANTA, sc-8017, mouse), diluted at 1:1000; Anti-PSMD11 antibody (ABclonal, A15306, rabbit), diluted at 1:500; Anti-PSMB5 antibody (ABclonal, A1975, rabbit), diluted at 1:1000; Anti-Proteasome 20S alpha + beta antibody (Abcam, ab22673, rabbit), diluted at 1:1000; Anti-GAPDH antibody (ABclonal, A19056, rabbit), diluted at 1:5000; Anti-Calnexin antibody (proteintech, 10427-2-AP, rabbit), diluted at 1:10,000; HRP-linked anti-rabbit IgG (CST, 7074, goat), diluted at 1:10,000; and HRP-linked anti-mouse IgG (CST, 7076, horse), diluted at 1:10,000.

Techniques: Activity Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Proteasome-Enriched hPPSCs-Derived EVs Attenuate Hypoxic Injury in Endothelial Cells via Proteasome-Mediated HIF-1α Degradation

doi: 10.3390/ijms27157013

Figure Lengend Snippet: Proteasomes in hPPSCs-EVs suppress HIF-1α expression and ubiquitinated protein accumulation induced by CoCl 2 . ( A ) Representative micrographs of hUVECs in different treatment groups. Groups were set as follows: control hUVECs cultured in serum-free medium; CoCl 2 hUVECs processed with 300 μM CoCl 2 for 72 h; CoCl 2 +human umbilical cord mesenchymal stem cells (hUCMSCs)-EVs hUVECs treated with 300 μM CoCl 2 and 75 μg/mL hUCMSCs-EVs for 72 h; and CoCl 2 +hPPSCs-EVs hUVECs treated with 300 μM CoCl 2 and 75 μg/mL hPPSCs-EVs for 72 h (magnification, 200×; scale, 50 μm). ( B ) Quantitative analysis of the number of adherent endothelial cells, n = 3, p < 0.05. ( C ) The protein expression levels of HIF-1α, 20S proteasome, PSMD11, PSMB5, and ubiquitinated proteins. ( D ) Quantitative analysis of HIF-1α, 20S proteasome, PSMD11, and PSMB5 protein. n = 3, p < 0.05.

Article Snippet: The antibodies employed for Western blot in this study are listed below: Anti-ALIX antibody (Abcam, ab275377, rabbit, Cambridge, MA, USA), diluted at 1:1000; Anti-CD81 antibody (ABclonal, A5270, rabbit, Woburn, MA, USA), diluted at 1:1000; Anti-CD63 antibody (ABclonal, A19023, rabbit), diluted at 1:1000; Anti-HIF-1α antibody (ABclonal, A16873, rabbit), diluted at 1:1000; Anti-Ubiquitin (P4D1) antibody (SANTA, sc-8017, mouse), diluted at 1:1000; Anti-PSMD11 antibody (ABclonal, A15306, rabbit), diluted at 1:500; Anti-PSMB5 antibody (ABclonal, A1975, rabbit), diluted at 1:1000; Anti-Proteasome 20S alpha + beta antibody (Abcam, ab22673, rabbit), diluted at 1:1000; Anti-GAPDH antibody (ABclonal, A19056, rabbit), diluted at 1:5000; Anti-Calnexin antibody (proteintech, 10427-2-AP, rabbit), diluted at 1:10,000; HRP-linked anti-rabbit IgG (CST, 7074, goat), diluted at 1:10,000; and HRP-linked anti-mouse IgG (CST, 7076, horse), diluted at 1:10,000.

Techniques: Expressing, Control, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Proteasome-Enriched hPPSCs-Derived EVs Attenuate Hypoxic Injury in Endothelial Cells via Proteasome-Mediated HIF-1α Degradation

doi: 10.3390/ijms27157013

Figure Lengend Snippet: Proteasomes in hPPSCs-EVs inhibit MG-132-induced HIF-1α expression. ( A ) Representative micrographs of hUVECs in different treatment groups. Groups were set as follows: control hUVECs cultured in serum-free medium; MG-132 hUVECs subjected to 0.25 μM MG-132 for 72 h; MG-132+hUCMSCs-EVs hUVECs treated with 0.25 μM MG-132 and 75 μg/mL hUCMSCs-EVs for 72 h; and MG-132+hPPSCs-EVs hUVECs treated with 0.25 μM MG-132 and 75 μg/mL hPPSCs-EVs for 72 h (magnification, 200×; scale, 50 μm). ( B ) Quantitative analysis of the number of adherent endothelial cells, n = 3, p < 0.05. ( C ) The protein expression levels of HIF-1α, 20S proteasome, PSMD11, PSMB5, and ubiquitinated proteins. ( D ) Quantitative analysis of HIF-1α, 20S proteasome, PSMD11, and PSMB5 protein. n = 3, p < 0.05.

Article Snippet: The antibodies employed for Western blot in this study are listed below: Anti-ALIX antibody (Abcam, ab275377, rabbit, Cambridge, MA, USA), diluted at 1:1000; Anti-CD81 antibody (ABclonal, A5270, rabbit, Woburn, MA, USA), diluted at 1:1000; Anti-CD63 antibody (ABclonal, A19023, rabbit), diluted at 1:1000; Anti-HIF-1α antibody (ABclonal, A16873, rabbit), diluted at 1:1000; Anti-Ubiquitin (P4D1) antibody (SANTA, sc-8017, mouse), diluted at 1:1000; Anti-PSMD11 antibody (ABclonal, A15306, rabbit), diluted at 1:500; Anti-PSMB5 antibody (ABclonal, A1975, rabbit), diluted at 1:1000; Anti-Proteasome 20S alpha + beta antibody (Abcam, ab22673, rabbit), diluted at 1:1000; Anti-GAPDH antibody (ABclonal, A19056, rabbit), diluted at 1:5000; Anti-Calnexin antibody (proteintech, 10427-2-AP, rabbit), diluted at 1:10,000; HRP-linked anti-rabbit IgG (CST, 7074, goat), diluted at 1:10,000; and HRP-linked anti-mouse IgG (CST, 7076, horse), diluted at 1:10,000.

Techniques: Expressing, Control, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Proteasome-Enriched hPPSCs-Derived EVs Attenuate Hypoxic Injury in Endothelial Cells via Proteasome-Mediated HIF-1α Degradation

doi: 10.3390/ijms27157013

Figure Lengend Snippet: The hPPSCs-EVs proteasomes degrade HIF-1α induced by both CoCl 2 and MG-132. ( A ) Representative micrographs of hUVECs in different treatment groups. Groups were set as follows: control hUVECs cultured in serum-free medium; CoCl 2 +MG-132 hUVECs treated with 300 μM CoCl 2 and 0.25 μM MG-132 for 72 h; CoCl 2 +MG-132+hUCMSCs-EVs hUVECs treated with 300 μM CoCl 2 , 0.25 μM MG-132, and 75 μg/mL hUCMSCs-EVs for 72 h; and CoCl 2 +MG-132+hPPSCs-EVs hUVECs treated with 300 μM CoCl 2 , 0.25 μM MG-132, and 75 μg/mL hPPSCs-EVs for 72 h. (magnification: 200×; scale bar: 50 μm) ( B ) Adherent endothelial cell count quantitation, n = 3, p < 0.05. ( C ) The protein expression levels of HIF-1α, 20S proteasome, PSMD11, PSMB5, and ubiquitinated proteins. ( D ) Quantitative analysis of HIF-1α, 20S proteasome, PSMD11, and PSMB5 protein. n = 3, p < 0.05.

Article Snippet: The antibodies employed for Western blot in this study are listed below: Anti-ALIX antibody (Abcam, ab275377, rabbit, Cambridge, MA, USA), diluted at 1:1000; Anti-CD81 antibody (ABclonal, A5270, rabbit, Woburn, MA, USA), diluted at 1:1000; Anti-CD63 antibody (ABclonal, A19023, rabbit), diluted at 1:1000; Anti-HIF-1α antibody (ABclonal, A16873, rabbit), diluted at 1:1000; Anti-Ubiquitin (P4D1) antibody (SANTA, sc-8017, mouse), diluted at 1:1000; Anti-PSMD11 antibody (ABclonal, A15306, rabbit), diluted at 1:500; Anti-PSMB5 antibody (ABclonal, A1975, rabbit), diluted at 1:1000; Anti-Proteasome 20S alpha + beta antibody (Abcam, ab22673, rabbit), diluted at 1:1000; Anti-GAPDH antibody (ABclonal, A19056, rabbit), diluted at 1:5000; Anti-Calnexin antibody (proteintech, 10427-2-AP, rabbit), diluted at 1:10,000; HRP-linked anti-rabbit IgG (CST, 7074, goat), diluted at 1:10,000; and HRP-linked anti-mouse IgG (CST, 7076, horse), diluted at 1:10,000.

Techniques: Control, Cell Culture, Cell Characterization, Quantitation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Andrographolide targets syndecan4 to impair its interaction with syntenin and inhibits the biogenesis of small extracellular vesicles

doi: 10.1016/j.jbc.2026.111182

Figure Lengend Snippet: MG132 inhibits the degradation of SDC4-CTF. A and B , Western blot of HCT116 cells treated with 0.5, 1, 2.5, or 5 μM MG132 for 12 h, or with 2.5 μM MG132 for 0 to 12 h. C , Western blot of full-length SDC1 and SDC4 in HCT116 cells treated with MG132 (0.5–5 μM, 12 h). D and E , immunofluorescence detection and quantification of HCT116 cells transfected with SDC1-GFP or SDC4-GFP, with/without 5 μM MG132 (4 h; n = 6). F , Western blot of multiple colorectal cancer cell lines treated with 10 μM MG132 (12 h). G , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with 0.1, 0.5, or 1 μM proteasome inhibitors (Carfilzomib, Ixazomib, Bortezomib) for 24 h. H , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells treated with PD150606 (2, 5, 10 μM) for 12 h. I , quantification of DQ-BSA fluorescence with MG132 or Earle’s balanced salt solution (EBSS) treatment (n = 4). J , quantification of lysosomal activity using LysoTracker with MG132 or EBSS treatment (n = 4). K , Western blot of SDC1-CTF and SDC4-CTF in HCT116 cells with siRNA knockdown of proteasome subunits PSMD14, PSMD2, USP14, PSMB5, PSMA6. Data: mean ± SD (≥3 experiments). Statistics: unpaired two-tailed t test (∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). SDC4, syndecan4; CTF, C-terminal transmembrane (TM) fragment; EBSS, Earle’s balanced salt solution.

Article Snippet: SDC1 (12,922) for SDC1-CTF detection, PSMB5 (12,919), Phospho-p65 (3033), Rab5 (3547), and GAPDH (5174) antibodies were obtained from Cell Signaling Technology.

Techniques: Western Blot, Immunofluorescence, Transfection, Fluorescence, Activity Assay, Knockdown, Two Tailed Test

Journal: iScience

Article Title: Ubiquitination and degradation of CD47 enhances macrophage phagocytosis of hemolytic erythrocytes

doi: 10.1016/j.isci.2025.114499

Figure Lengend Snippet: Changes in the major protein components of the proteasome during hemolysis (A) Western blot analysis of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in control and hemolysis groups. (B and C) Quantification of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in the RBC membrane (B) and cytoplasm (C) ( n = 3). (D) Representative immunofluorescence images of proteasomal subunits (PSME1/2, PSMB5/6/7) of hemolytic RBCs ( n = 6). Scale bars = 10 μm. (E) Schematic illustration of the animal model of immune hemolysis. (F and G) Immunofluorescence of proteasomal subunits (PSME1/2, PSMB5/6/7) in hemolytic mouse RBCs (F) and AIHA patient RBCs (G) ( n = 6). Scale bars = 10 μm. (H–J) Statistical analysis of caspase-like activity (H), trypsin-like activity (I), and chymotrypsin-like activity (J) of the membrane proteins after hemolysis ( n = 6). Data were analyzed by Student’s t test (two groups) or one-way ANOVA with Tukey’s test (multiple groups) and are expressed as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns = no significance.

Article Snippet: The primary antibodies utilized in this procedure were CD47 (Santa Cruz Biotechnology, Cat# sc-12730), MARCH1 (HUABIO Biotechnology, Cat# ER63906), PSME1 (Abcam, Cat# ab186832), PSME2 (Abcam, Cat# ab183727), PSMB5 (BOSTER, Cat# A03418-1), PSMB6 (ABclonal Technology, Cat# A4053), PSMB7 (BOSTER, Cat# A08095-1), and UBQLN1 (Proteintech Group, Cat# 22126-1-AP).

Techniques: Western Blot, Expressing, Control, Membrane, Immunofluorescence, Animal Model, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: α‑Aminoboronic Acid Moieties in Boro Dipeptides Modulate Proteasome Subunit Selectivity and Provide Access to Compounds with Potent Anticancer and Anti-Inflammatory Activity

doi: 10.1021/acs.jmedchem.5c02548

Figure Lengend Snippet: Effect of compounds 5b , 5c , and 5j – l , as well as subunit selective control compounds on the secretion of cytokines in LPS-stimulated PBMCs. All compounds were tested at 250 nM. The cells were pretreated for 1 h with inhibitors, followed by the addition of LPS (1 μg/mL). As the negative control (designated by ‘DMSO’ in figure legends), cells were treated only with DMSO, followed by the addition of LPS (1 μg/mL). The concentrations of cytokines were determined in the supernatants after additional 24 h treatment. The results are represented as means ± SD of four independent experiments ( N = 4). Statistical significance between untreated controls versus treated was calculated using one-way ANOVA post hoc Dunnett’s test. A p -value of less than 0.05 was considered significant (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05).

Article Snippet: Membranes were blocked with 5% BSA in 1 × TTBS for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against: β5i (1:1000; #13726), β5c (1:1000; #12919), and β1c (1:1000; #13267; all from Cell Signaling Technology), β1i (1:1000; #ab187645; all from Abcam), β2c (1:1000, #ab154745), and β2i (1:1000, #ab183506), and β-actin (1:5000; #A5316; SigmaPrestige).

Techniques: Control, Negative Control