rabbit polyclonal anti mmp 9 antibody  (Proteintech)

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: MMP-9 , a factor that promotes Vasculogenic mimicry, is highly expressed in CC and is associated with poor prognosis. (A) CC database of TCGA was used to analyze key factors associated with VM. (B) Association of Sox2 expression with overall survival in CC (log-rank test). (C) Association of MMP-9 expression with overall survival in CC (log-rank test). (D) Panoramic scans after immunohistochemical detection of MMP-9 and H&E staining in samples from cancerous and paracancerous tissues from subjects with CC. Scale bar, 50 µm. Original magnification, ×20. (E) Protein levels of MMP-9 in 20 paired samples, with the MMP-9 level in CC tissue expressed compared with that in the paired normal tissue. (F) Expression levels of MMP-9 mRNA in 44 paired CC and paracancerous tissues, with MMP-9 expression in CC tissue expressed compared with that in the paired normal tissue. (G) Comparison of the average expression levels of MMP-9 mRNA in CC tissues compared with paracancerous tissues. (H) HeLa and SiHa cells were incubated under hypoxia (0.1% O 2 ) and proteins collected at 24, 48 and 72 h for western blotting of ALDH1, EPHA2, MMP-9 and GAPDH. ImageJ was used to semi-quantify western blotting signals from HeLa (I) and SiHa (J) cells. GAPDH served as an internal reference. *P<0.05, **P<0.01 and ***P<0.001. MMP-9, matrix metalloproteinase 9; VM, vasculogenic mimicry; ALDH1, aldehyde dehydrogenase 1; EPHA2, ephrin type-A receptor 2; TCGA, The Cancer Genome Atlas; Sox2, SRY-box transcription factor 2; CC, cervical cancer; CESC, cervical squamous cell carcinoma.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Incubation, Western Blot

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: NSUN2 promotes Vasculogenic mimicry, invasion and migration of CC cells under hypoxic conditions. (A) Expression levels of NSUN2 in CC cells lines (HeLa, SiHa, CaSki, C33A and HT-3) and in a normal cervical cell line (HaCaT). (B) RT-qPCR was used to determine relative expression levels of NSUN2 mRNA in HeLa and SiHa cells after transfection of shRNAs and incubation under hypoxia for 24 h. (C) Relative expression levels of MMP-9 mRNA in HeLa and SiHa cells transfected with the NSUN2 -interfering plasmid and incubated under hypoxia for 24 h. (D) Dot blot assay analysis of m 5 C expression levels in HeLa and SiHa cells after 48 h of hypoxia culture following transfection with NSUN2 knockdown plasmids. (E) Semi-quantitative analysis of dot blot results in HeLa cells. (F) Semi-quantitative analysis of dot blot results in SiHa cells. (G) Western blotting was used to investigate NSUN2 and MMP-9 protein levels in HeLa and SiHa cells transfected with the NSUN2 -interfering plasmid and incubated under hypoxia for 48 h. ImageJ was used to semi-quantify western blotting bands for (H) NSUN2 and (I) MMP-9 protein levels. (J) 2D tube-forming assays of HeLa and SiHa cells transfected with an NSUN2 -interfering plasmid and incubated under hypoxia. Scale bar, 100 µm. Original magnification, ×4. (K) Assay displayed in panel (J) was quantified using Image Pro. (L) Invasion assay of HeLa and SiHa cells transfected with a control plasmid, shNSUN2-2 or shNSUN2-3 or with shNSUN2 and pcDNA MMP-9. Scale bar, 200 µm. Original magnification, ×10. (M) Assay displayed in panel was quantified using Image Pro. (N) Migration assay of HeLa and SiHa cells transfected with a control plasmid, shNSUN2-2 or shNSUN2-3 or with shNSUN2 and pcDNA MMP-9. Scale bar, 200 µm. Original magnification, ×10. (O) Assay displayed in panel (N) was quantified using Image Pro. *P<0.05, **P<0.01 and ***P<0.001. VM, Vasculogenic mimicry; CC, cervical cancer; IHC, immunohistochemistry; NSUN2, NOP2/Sun RNA methyltransferase 2; m 5 C, 5-methylcytidine; RT-qPCR, reverse transcription-quantitative PCR; IHC, immunohistochemistry; CaSki, human cervical cancer cell line with intestinal metastasis; C33A, human cervical cancer cell line; HaCaT, human skin keratinocytes cell line; HeLa, human cervical cancer cell line; HT-3, human cervical cancer cell line; MMP9, matrix metalloproteinase 9; SiHa, human cervical squamous cell line; pcDNA, plasmid cloning DNA; shRNA, short hairpin RNA; NC, negative control.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Migration, Expressing, Quantitative RT-PCR, Transfection, Incubation, Plasmid Preparation, Dot Blot, Knockdown, Quantitative Dot Blot, Western Blot, Invasion Assay, Control, Immunohistochemistry, Reverse Transcription, Real-time Polymerase Chain Reaction, Cloning, shRNA, Negative Control

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: NSUN2 increases the stability of MMP-9 mRNA. (A) A positive correlation was observed between NSUN2 and MMP-9 mRNA expression levels in 44 pairs of samples from subjects with CC. Enrichment of the m 5 C modification of MMP-9 mRNA in HeLa (B) and SiHa (C) Cells were measured with anti-m 5 C methylated-RNA IP assays. Interaction of NSUN2 with MMP-9 mRNA in (D) HeLa and (E) SiHa cells was measured with anti- NSUN2 RNA IP assays. Stability of MMP-9 mRNA after interference with and overexpression of NSUN2 was measured in (F) HeLa and (G) SiHa cells. (H) A model illustrating the proposed mechanism by which NSUN2-mediated stabilization of MMP-9 mRNA promotes Vasculogenic mimicry in CC. NSUN2, NOP2/Sun RNA methyltransferase 2; m 5 C, 5-methylcytidine; pcDNA, plasmid cloning DNA; shRNA, short hairpin RNA; HeLa, human cervical cancer cell line; MMP9, matrix metalloproteinase 9; SiHa, human cervical squamous cell line; IP, immunoprecipitation; CC, cervical cancer; NC, negative control.

Article Snippet: The tissue samples were treated with PH9.0 EDTA repair solution for antigen retrieval and then treated with a rabbit polyclonal anti-CD31 antibody (1:2,000; cat. no. AB76533; Abcam), a rabbit polyclonal anti-NSUN2 antibody (1:200; cat. no. AB259941; Abcam) or a rabbit polyclonal anti-MMP-9 antibody (1:200; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology).

Techniques: Expressing, Modification, Methylation, Over Expression, Plasmid Preparation, Cloning, shRNA, Immunoprecipitation, Negative Control

Journal: iScience

Article Title: Changes in the microglial phenotype drive neuroinflammation independent of systemic inflammation in the acute stage of heatstroke

doi: 10.1016/j.isci.2026.115254

Figure Lengend Snippet: Time course of functional damage to the intestinal barrier and blood-brain barrier during the acute stage of heatstroke The mice were exposed to 41.2 ± 0.5°C ambient temperature until their rectal temperature reached 42.4°C, and then they were allowed to recover at an ambient temperature of 25 ± 0.5°C for the indicated times. (A) The concentration of FD-4 in serum was measured after its oral administration. (B) Serum endotoxin levels. (C) EB was injected into the mice via the tail vein after the onset of heatstroke, and representative images of EB leakage was shown. (D) Quantitative analysis of EB concentrations in brain tissues. (E) Expression of MMP-2 in the cerebral cortex at 1 h, 6 h, and 24 h post heatstroke. (F) Expression of MMP-9 in the cerebral cortex at 1 h, 6 h, and 24 h post heatstroke. The data are expressed as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, vs. Con.

Article Snippet: The membrane was further incubated overnight at 4°C with primary antibodies against MMP9 (TA326652; ORIGENE, USA), MMP2 (TA326260; ORIGENE, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 2118L; CST, USA).

Techniques: Functional Assay, Concentration Assay, Injection, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: Bioinformatics analysis of AFP and MICA/B expression and function, and immunohistochemistry assay to detect AFP and MICA/B expression in HCC tissues. (A) Volcano plot demonstrating differentially expressed genes in HCC RNA‐Seq data in TCGA data; (B) Box plot demonstrating the expression level of AFP, MICA and MICB in HCC tissues and adjacent normal liver tissues in TCGA data; (C) Volcano plot demonstrating differentially expressed genes in HCC RNA‐Seq data in GEO data; (D) Box plot demonstrating AFP, MICA and MICB expression levels in HCC and adjacent normal liver tissues in GEO data; (E) Expression level of AFP and MICA/B in HCC tissues and adjacent normal liver tissues; (F) PPI network graphs of AFP and MICA/B; (G) KEGG enrichment analysis of co‐expression gene; (H) Lollipop plot demonstrating the correlation between AFP and immune cells; (I) Correlation scatter plot of AFP and NK cells; (J) Correlation scatter plot of AFP and MMP9, ADAM17, MMP14 and ADAM10. Scale length is 100 μm; * p < 0.05; ** p < 0.01; *** p < 0.001. ADAM10, de‐integrin‐metalloproteinase 10; ADAM17, de‐integrin‐metalloproteinase 17; AFP, Alpha‐fetoprotein; GEO, Gene Expression Omnibus; KEGG, Kyoto Genome and Genome Database; MICA/B, Major histocompatibility complex class I chain‐related proteins A and B; MMP14, matrix metalloproteinase 14; MMP9, matrix metalloproteinase 9; PPI, protein–protein interactions; RNA‐Seq, RNA sequencing; TCGA, the Cancer Genome Atlas.

Article Snippet: After washing for 10 min, the PVDF membrane was blocked with a protein‐free rapid blocking buffer (Cat #PS108, Epizyme, China) and incubated with the primary antibodies AFP (Cat #ab169552, Abcam, USA) at a dilution of 1:1000, MICA/B (Cat #ab224702, Abcam, USA) at a dilution of 1:1000, MMP9 (Cat #13667, Cell Signalling Technology, USA) at a dilution of 1:1500, PI3K (Cat #ab302958, Abcam, USA) at a dilution of 1:1000, AKT (Cat #ab8805, Abcam, USA) at a dilution of 1:500, p‐AKT (Cat #4060, Cell Signalling Technology, USA) at a dilution of 1:500, GAPDH (Cat #10494‐1‐AP, Proteintech, China) at a dilution of 1:10,000, and ATP1A1 (Cat #14418‐1‐AP, Proteintech, China) at a dilution of 1:10,000 at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, RNA Sequencing, Gene Expression, Immunopeptidomics, Protein-Protein interactions

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: The effect of AFP on the expression of MMP9 and MICA/B shedding on the membrane of HCC cells. (A) Western blotting experiment was performed to detect the expression levels of MMP9; (B) Grey scale values of MMP9 expression were calculated using Image J software and then statistically analysed and plotted; (C) qRT‐PCR experiment was used to check MMP9 mRNA expression levels; (D) Western blotting was performed to detect the expression levels of sMICA/B in HLE, HLE‐NC, HLE‐AFP cells; (E) Western blotting was performed to detect the expression levels of sMICA/B in HuH‐7, HuH‐7‐NC, HuH‐7‐shAFP cells; (F) ELISA were performed to detect the content of sMICA/B in HCC cell supernatants; (G) CCK‐8 experiments were performed to detect the IC50 of GM6001 and TAPI‐1; (H) Detection of MICA/B expression in HCC cells while treated with GM6001 and TAPI‐1 by flow cytometry; (I) Prism GraphPad(10.0) for statistical analysis and graphing of flow cytometry results; (J) Detection of sMICA/B in HCC cells supernatants while treated with GM6001 and TAPI‐1 by ELISA; (K) Western blotting experiment was performed to detect the expression levels of PI3K, AKT, p‐AKT and MMP9; (L) Grey scale values of protein expression were calculated using Image J software and then statistically analysed and plotted. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. The picture represents at least three repetitions of the experiment. AKT, protein kinase B; GM6001 and TAPI‐1, MMP9 inhibitors; MMP9, matrix metallopeptidase 9; p‐AKT, phospho‐protein kinase B; PI3K, phosphatidylinositol‐3‐kinase; sMICA/B, soluble major histocompatibility complex class I chain‐related proteins A and B.

Article Snippet: After washing for 10 min, the PVDF membrane was blocked with a protein‐free rapid blocking buffer (Cat #PS108, Epizyme, China) and incubated with the primary antibodies AFP (Cat #ab169552, Abcam, USA) at a dilution of 1:1000, MICA/B (Cat #ab224702, Abcam, USA) at a dilution of 1:1000, MMP9 (Cat #13667, Cell Signalling Technology, USA) at a dilution of 1:1500, PI3K (Cat #ab302958, Abcam, USA) at a dilution of 1:1000, AKT (Cat #ab8805, Abcam, USA) at a dilution of 1:500, p‐AKT (Cat #4060, Cell Signalling Technology, USA) at a dilution of 1:500, GAPDH (Cat #10494‐1‐AP, Proteintech, China) at a dilution of 1:10,000, and ATP1A1 (Cat #14418‐1‐AP, Proteintech, China) at a dilution of 1:10,000 at 4°C overnight.

Techniques: Expressing, Membrane, Western Blot, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Flow Cytometry, Immunopeptidomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: The effect of AFP on the expression of MMP9 and the membrane level of MICA/B in the NOD/SCID mouse tumour model. (A) Western blotting experiments were performed to detect the expression levels of AFP, MMP9 and MICA/B (MP); the bar chart on the right shows the grey value statistics of protein bands; (B) Immunohistochemistry experiments were performed to detect the expression levels of AFP, MMP9 and MICA/B. The scale length is 100 μm; the bar chart on the right shows the mean density value of proteins that were used for statistics. * p < 0.05; ** p < 0.01. MP, membrane protein.

Article Snippet: After washing for 10 min, the PVDF membrane was blocked with a protein‐free rapid blocking buffer (Cat #PS108, Epizyme, China) and incubated with the primary antibodies AFP (Cat #ab169552, Abcam, USA) at a dilution of 1:1000, MICA/B (Cat #ab224702, Abcam, USA) at a dilution of 1:1000, MMP9 (Cat #13667, Cell Signalling Technology, USA) at a dilution of 1:1500, PI3K (Cat #ab302958, Abcam, USA) at a dilution of 1:1000, AKT (Cat #ab8805, Abcam, USA) at a dilution of 1:500, p‐AKT (Cat #4060, Cell Signalling Technology, USA) at a dilution of 1:500, GAPDH (Cat #10494‐1‐AP, Proteintech, China) at a dilution of 1:10,000, and ATP1A1 (Cat #14418‐1‐AP, Proteintech, China) at a dilution of 1:10,000 at 4°C overnight.

Techniques: Expressing, Membrane, Western Blot, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: The influence of AFP and NKG2D‐neutralising antibodies on the cytotoxicity of NK‐92 cells to HCC cells. (A) Detection of NK‐92 cell surface markers by flow cytometry; (B) Detection of the cytotoxicity of NK‐92 cells to HCC cells by LDH release assay; (C) LDH release assay was used to detect the cytotoxicity of NK‐92 cells to HCC cells while treated with NKG2D neutralising antibodies; (D) LDH release assay was used to detect the cytotoxicity of NK‐92 cells to HCC cells while treated with MMP9 inhibitors (GM6001+TAPI‐1). * p < 0.05; ** p < 0.01; *** p < 0.001. LDH, lactate dehydrogenase; NKG2D, natural killer group 2 member D.

Article Snippet: After washing for 10 min, the PVDF membrane was blocked with a protein‐free rapid blocking buffer (Cat #PS108, Epizyme, China) and incubated with the primary antibodies AFP (Cat #ab169552, Abcam, USA) at a dilution of 1:1000, MICA/B (Cat #ab224702, Abcam, USA) at a dilution of 1:1000, MMP9 (Cat #13667, Cell Signalling Technology, USA) at a dilution of 1:1500, PI3K (Cat #ab302958, Abcam, USA) at a dilution of 1:1000, AKT (Cat #ab8805, Abcam, USA) at a dilution of 1:500, p‐AKT (Cat #4060, Cell Signalling Technology, USA) at a dilution of 1:500, GAPDH (Cat #10494‐1‐AP, Proteintech, China) at a dilution of 1:10,000, and ATP1A1 (Cat #14418‐1‐AP, Proteintech, China) at a dilution of 1:10,000 at 4°C overnight.

Techniques: Flow Cytometry, Lactate Dehydrogenase Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: Mechanism diagram of AFP promotes resistance to NK‐92 cytotoxicity in HCC cells by regulating MICA/B expression. AFP promotes the expression of MMP9 to shed MICA/B on the cell membrane of liver cancer, and the deciduous MICA/B from liver cancer cells binds to NKG2D on the cell membrane of NK cells, masking the NKG2D in NK cells to recognise MICA/B on the cell membrane of liver cancer cells, resulting in liver cancer cells escaping from the attack of NK cells. AFP, alpha‐fetoprotein; AKT, protein kinase B; HCC, hepatocellular carcinoma; MICA/B, major histocompatibility complex class I‐related chain A/B; MMP9, matrix metalloproteinases 9; NK‐92, natural killer‐92; NKG2D, natural killer group 2 member D; PI3K, phosphatidylinositol‐3‐kinase; sMICA/B, soluble MICA/B.

Article Snippet: After washing for 10 min, the PVDF membrane was blocked with a protein‐free rapid blocking buffer (Cat #PS108, Epizyme, China) and incubated with the primary antibodies AFP (Cat #ab169552, Abcam, USA) at a dilution of 1:1000, MICA/B (Cat #ab224702, Abcam, USA) at a dilution of 1:1000, MMP9 (Cat #13667, Cell Signalling Technology, USA) at a dilution of 1:1500, PI3K (Cat #ab302958, Abcam, USA) at a dilution of 1:1000, AKT (Cat #ab8805, Abcam, USA) at a dilution of 1:500, p‐AKT (Cat #4060, Cell Signalling Technology, USA) at a dilution of 1:500, GAPDH (Cat #10494‐1‐AP, Proteintech, China) at a dilution of 1:10,000, and ATP1A1 (Cat #14418‐1‐AP, Proteintech, China) at a dilution of 1:10,000 at 4°C overnight.

Techniques: Expressing, Membrane, Immunopeptidomics