anti calnexin  (Boster Bio)

Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs engineering boosts the production of ADSCs-EVs with superior yield and stability A. Schematic illustration of the high-efficiency extraction of extracellular vesicles (EVs) from adipose-derived stem cells (ADSCs) using nanosecond pulsed electric fields (NsPEFs). B. Representative transmission electron microscopy (TEM) images of isolated Ctrl-ADSCs-EVs and NsPEFs-ADSCs-EVs, showing characteristic cup-shaped morphology and bilayer membrane (scale bars: 150 nm and 75 nm). C. Nanoparticle tracking analysis (NTA) showing the particle size distribution of EVs (n = 3). D. Western blot (WB) analysis confirming the positive expression of EV-specific markers (CD81, CD63, TSG101) and the absence of the negative markers (Calnexin, Histone H3, LaminA/C). Quantification is shown on the right (n = 3). E. The particle concentration of EVs. F. NsPEFs stimulation significantly enhanced both yield and protein output compared to Ctrl-ADSCs-EVs. G. Zeta potential measurement indicating colloidal stability (n = 3). H. Purity assessment expressed as the particle-to-protein ratio ( × 10 9 particles/μg). I. Viability of cells post-NsPEFs-ADSCs-EVs treatment assessed by trypan blue exclusion assay (scale bar: 1.7 mm). Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001; ns: not significant.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Extraction, Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Membrane, Western Blot, Expressing, Concentration Assay, Zeta Potential Analyzer, Trypan Blue Exclusion Assay, Two Tailed Test

Journal: Bioactive Materials

Article Title: NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties

doi: 10.1016/j.bioactmat.2026.01.006

Figure Lengend Snippet: NsPEFs-ADSCs-EVs induce RSPO3 secretion via an ITGA4/PI3K/Akt-dependent mechanism. A- D.Proteomic profiling identifies ITGA4 as a key mediator linking NsPEFs-ADSCs-EVs to RSPO3. (A). Significantly enriched proteins in NsPEFs-ADSCs-EVs using proteomic analysis (n = 3). (B, C). Gene Ontology and KEGG pathway enrichment analyses of proteins in NsPEFs-ADSCs-EVs, highlight integrin binding, cell adhesion and PI3K-Akt signaling. (D). The protein-protein interaction network integrating RSPO3 with top enriched EV proteins, reveals a potential functional link with ITGA4. E- I.EV-surface ITGA4 is essential for chondrocyte targeting and RSPO3 induction. (E). The schematic hypothesizes the ITGA4-initiated signaling axis related to RSPO3 secretion. (F) qPCR analysis shows that the increased transcriptional level of Rspo3 in chondrocytes treated with NsPEFs-ADSCs-EVs is inhibited by an ITGA4-neutralizing antibody (Trosunilimab) (n = 6). (G). qPCR validation of Itga4 knockdown efficiency in ADSCs (n = 6). (H). Western blot analysis confirms the successful generation of ITGA4-deficient EVs (NsPEFs-EVs-ITGA4-KD) from Itga4 -knockdown ADSCs, while maintaining EV purity (CD63 + /Calnexin − ) (n = 3). (I) Cellular uptake of DiR-labeled NsPEFs-ADSCs-EVs-ITGA4-KD by chondrocyte is significantly impaired compared to that of NsPEFs-ADSCs-EVs-NC. Quantification of fluorescence intensity is shown (scale bar: 36.8 μm; n = 3). J-L.ITGA4 initiates RSPO3 expression through the PI3K/Akt pathway. (J). Western blot analysis of Akt phosphorylation (p-Akt) and RSPO3 in chondrocytes treated with the indicated EVs (n = 3). (K). qPCR analysis of Rspo3 confirms that ITGA4-deficient EVs fail to induce RSPO3 expression (n = 6). (L). Pharmacological inhibition of PI3K (LY294002) or Akt (MK-2206) abolishes NsPEFs-ADSCs-EVs-induced Rspo3 upregulation in chondrocytes (n = 6). Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies used and the dilution ratios were as follows:Anti-INOS (1:800, Cohesion), Anti-Arginase 1 (1:800, BOSTER), Anti-LRP6 (1:800, BOSTER), Anti-Beta-catenin (1:800, BOSTER), Anti-CD163 (1:800, Abclonal), Anti-CD86 (1:800, BOSTER), Anti-LGR4 (1:800, Abclonal), Anti-IL-1β (1:800, BOSTER), Anti-IL-10 (1:1000, Bioss), Anti-MMP13 (1:800, BOSTER), Anti-COL2A1 (1:800, BOSTER), Anti-Histone H3 (1:1000, Nature Biosciences), Anti-Lamin A/C (1:1000, Nature Biosciences), Anti-Akt (1:1000, Nature Biosciences), Anti-pAkt (1:1000, Nature Biosciences), Anti-RSPO3 (1:1000, Abcam), Anti-CD63(1:800, BOSTER), Anti-CD81(1:800, BOSTER), Anti-TSG101(1:800, BOSTER), Anti-Calnexin(1:800, BOSTER).

Techniques: Binding Assay, Functional Assay, Biomarker Discovery, Knockdown, Western Blot, Labeling, Fluorescence, Expressing, Phospho-proteomics, Inhibition, Two Tailed Test

Journal: Molecular Medicine Reports

Article Title: Exosomal SphK1 from colorectal cancer cells promotes cancer cell migration and activates hepatic stellate cells

doi: 10.3892/mmr.2025.13438

Figure Lengend Snippet: Brief experimental process of the present study. Cell transfection technique was used to construct RKO cells with low expression of SphK1. Western blotting and PCR were used to verify the expression of SphK1 in RKO cells. The culture supernatant of SphK1(−)-RKO cells was collected, and the exosomes were extracted and identified by TEM, NTA and western blotting. The extracted exosomes were used to co-culture with RKO cells, and the expression of E-cadherin and vimentin was detected by western blotting. The migration ability of RKO cells was detected by Transwell assay. The extracted exosomes were also used to co-culture with LX-2 cells, and the expression levels of AKT, p-AKT, α-SMA, TNF-α and TGF-β were detected by western blotting. The LX-2 cell culture supernatant was extracted and used to culture RKO cells. CCK-8 was used to detect RKO cell viability, and western blotting was used to detect PCNA expression in RKO cells. In addition, LX-2 cells were pretreated with SC79 and treated with RKO exosomes, and the expression levels of AKT, p-AKT, α-SMA, TNF-α and TGF-β were detected by western blotting. α-SMA, α-smooth muscle actin; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis; WB, western blotting; SphK1, sphingosine kinase 1; PCNA, proliferating cell nuclear antigen; CCK-8, Cell Counting Kit-8; p-, phosphorylated.

Article Snippet: The primary antibodies used were as follows: GAPDH polyclonal antibody (cat. no. 10494-1-AP; 1:4,000), SphK1 polyclonal antibody (cat. no. 10670-1-AP; 1:1,000), TSG101 polyclonal antibody (cat. no. 28283-1-AP; 1:4,000), vimentin polyclonal antibody (cat. no. 10366-1-AP; 1:2,000), CD81 monoclonal antibody (cat. no. 66866-1-Ig; 1:1,000), E-cadherin monoclonal antibody (cat. no. 60335-1-Ig; 1:4,000) and phosphorylated (p-)AKT (Ser473) monoclonal antibody (cat. no. 66444-1-Ig; 1:2,000) were purchased from Proteintech Group, Inc. α-SMA (cat. no. BM3902; 1:4,000) and AKT (cat. no. A00024-2; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. Calnexin (cat. no. HY-P80578; 1:1,000), proliferating cell nuclear antigen (PCNA; cat. no. HY-P80268; 1:2,000), TGF-β1 (cat. no. HY-P80521; 1:500) and TNF-α (cat. no. HY-P80914; 1:1,000) antibodies were purchased from MedChemExpress.

Techniques: Transfection, Construct, Expressing, Western Blot, Co-Culture Assay, Migration, Transwell Assay, Cell Culture, CCK-8 Assay, Transmission Assay, Electron Microscopy, Cell Counting

Journal: Molecular Medicine Reports

Article Title: Exosomal SphK1 from colorectal cancer cells promotes cancer cell migration and activates hepatic stellate cells

doi: 10.3892/mmr.2025.13438

Figure Lengend Snippet: CRC-derived exosomes regulate p-AKT to activate hepatic stellate cells and promote the proliferation of CRC cells via exosomal SphK1. (A) PKH67-labeled RKO-exosomes were detected in LX-2 cells (magnification, ×200; scale bar, 50 µm). (B) Western blotting for AKT, p-AKT, α-SMA, TNF-α and TGF-β in LX-2 cells. (C) Western blotting for PCNA in RKO cells. (D) The viability rate of RKO cells was analyzed using a Cell Counting Kit-8 assay. *P<0.05. ns, not significant; exo, exosomes; CS, cell supernatant; α-SMA, α-smooth muscle actin; SphK1, sphingosine kinase 1; NC, negative control; Con, control; PCNA, proliferating cell nuclear antigen; p-, phosphorylated; CRC, colorectal cancer.

Article Snippet: The primary antibodies used were as follows: GAPDH polyclonal antibody (cat. no. 10494-1-AP; 1:4,000), SphK1 polyclonal antibody (cat. no. 10670-1-AP; 1:1,000), TSG101 polyclonal antibody (cat. no. 28283-1-AP; 1:4,000), vimentin polyclonal antibody (cat. no. 10366-1-AP; 1:2,000), CD81 monoclonal antibody (cat. no. 66866-1-Ig; 1:1,000), E-cadherin monoclonal antibody (cat. no. 60335-1-Ig; 1:4,000) and phosphorylated (p-)AKT (Ser473) monoclonal antibody (cat. no. 66444-1-Ig; 1:2,000) were purchased from Proteintech Group, Inc. α-SMA (cat. no. BM3902; 1:4,000) and AKT (cat. no. A00024-2; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. Calnexin (cat. no. HY-P80578; 1:1,000), proliferating cell nuclear antigen (PCNA; cat. no. HY-P80268; 1:2,000), TGF-β1 (cat. no. HY-P80521; 1:500) and TNF-α (cat. no. HY-P80914; 1:1,000) antibodies were purchased from MedChemExpress.

Techniques: Derivative Assay, Labeling, Western Blot, Cell Counting, Negative Control, Control

Journal: Molecular Medicine Reports

Article Title: Exosomal SphK1 from colorectal cancer cells promotes cancer cell migration and activates hepatic stellate cells

doi: 10.3892/mmr.2025.13438

Figure Lengend Snippet: Effect of SC79 on the activation of hepatic stellate cells. (A) Western blotting was used to examine the levels of AKT and p-AKT in LX-2 cells treated with SC79. (B) Western blotting was used to examine the levels of AKT, p-AKT, α-SMA, TNF-α and TGF-β in SphK1(−)-RKO exo + LX-2 cells treated with SC79. AKT and p-AKT isoforms exist and the antibodies could not be distinguished, so double bands appeared. *P<0.05. ns, not significant; exo, exosomes; α-SMA, α-smooth muscle actin; SphK1, sphingosine kinase 1; p-, phosphorylated.

Article Snippet: The primary antibodies used were as follows: GAPDH polyclonal antibody (cat. no. 10494-1-AP; 1:4,000), SphK1 polyclonal antibody (cat. no. 10670-1-AP; 1:1,000), TSG101 polyclonal antibody (cat. no. 28283-1-AP; 1:4,000), vimentin polyclonal antibody (cat. no. 10366-1-AP; 1:2,000), CD81 monoclonal antibody (cat. no. 66866-1-Ig; 1:1,000), E-cadherin monoclonal antibody (cat. no. 60335-1-Ig; 1:4,000) and phosphorylated (p-)AKT (Ser473) monoclonal antibody (cat. no. 66444-1-Ig; 1:2,000) were purchased from Proteintech Group, Inc. α-SMA (cat. no. BM3902; 1:4,000) and AKT (cat. no. A00024-2; 1:1,000) antibodies were purchased from Wuhan Boster Biological Technology, Ltd. Calnexin (cat. no. HY-P80578; 1:1,000), proliferating cell nuclear antigen (PCNA; cat. no. HY-P80268; 1:2,000), TGF-β1 (cat. no. HY-P80521; 1:500) and TNF-α (cat. no. HY-P80914; 1:1,000) antibodies were purchased from MedChemExpress.

Techniques: Activation Assay, Western Blot