thrombospondin motif 5 adamts 5 primary antibody  (Boster Bio)

Journal: Aging (Albany NY)

Article Title: Lei’s formula attenuates osteoarthritis mediated by suppression of chondrocyte senescence via the mTOR axis: in vitro and in vivo experiments

doi: 10.18632/aging.205582

Figure Lengend Snippet: LSF regulates chondrocyte anabolic-catabolic homeostasis and inhibits chondrocyte senescence. ( A – D ) Western blotting assessed the expression of Col2, Adamts5 and P16 proteins in chondrocytes. ( E – J ) qRT-PCR was performed to detect the expression of Col2, Sox9, MMP13, Adamts5, P16 and P21 in chondrocytes. ( K ) Cellular senescence was assessed by SA-β-gal staining. All data represent mean ± SD. Compared to the control group, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared to the H 2 O 2 -stimulated group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared to the same concentration of blank serum group, *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: Primary antibodies against CDKN2A/p16INK4a (1:100) were purchased from Zenbio (Chengdu, China), and the primary antibody against Adamts5 (1:50) was acquired from HUABIO (Hangzhou, China).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Lei’s formula attenuates osteoarthritis mediated by suppression of chondrocyte senescence via the mTOR axis: in vitro and in vivo experiments

doi: 10.18632/aging.205582

Figure Lengend Snippet: Activation of mTOR exacerbates H 2 O 2 -stimulated chondrocyte senescence and anabolic catabolic disorders that can be reversed by LSF. ( A ) Western blotting for protein expression of Col2, Adamts5, P16 and mTOR in chondrocytes processed with H 2 O 2 (200 μmol/L) and MHY1485 (10 μmol/L). ( B – E ) Quantitative results of Western blotting of the 5 groups of proteins. ( F – M ) Expression of Col2, Sox9, MMP13, Adamts5, P16, P21, mTOR, and S6 in chondrocytes processed with H 2 O 2 (200 μmol/L) and MHY1485 (10 μmol/L) was detected by qRT-PCR. ( N ) Cellular senescence was assessed by SA-β-gal staining. Compared to the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared to the H 2 O 2 -stimulated group, *P <0.05, *P < 0.01, ***P < 0.001, ****P<0.0001; compared to the H 2 O 2 -stimulated combined MHY1485 group, *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001.

Article Snippet: Primary antibodies against CDKN2A/p16INK4a (1:100) were purchased from Zenbio (Chengdu, China), and the primary antibody against Adamts5 (1:50) was acquired from HUABIO (Hangzhou, China).

Techniques: Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Staining

Journal: Aging (Albany NY)

Article Title: Lei’s formula attenuates osteoarthritis mediated by suppression of chondrocyte senescence via the mTOR axis: in vitro and in vivo experiments

doi: 10.18632/aging.205582

Figure Lengend Snippet: mTOR-SiRNA contributes to LSF suppression of H 2 O 2 -stimulated chondrocyte senescence and promotion of anabolism. ( A ) Westen blotting for protein expression of Col2, Adamts5, P16 and mTOR in lentivirus-transfected chondrocytes stimulated by H2O2 (200 μmol/L). ( B – E ) Quantitative results of Western blotting of the 4 groups of proteins. ( F – M ) Expression of Col2, Sox9, MMP13, Adamts5, P16, P21, mTOR, and S6 in lentivirus-transfected chondrocytes stimulated with H2O2 (200 μmol/L) was detected by qRT-PCR. ( N ) Cellular senescence was assessed by SA-β-gal staining. Compared to Con-SiRNA control group, ****P < 0.0001; compared to the H 2 O 2 -stimulated group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared to the mTOR-SiRNA group, *P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001.

Article Snippet: Primary antibodies against CDKN2A/p16INK4a (1:100) were purchased from Zenbio (Chengdu, China), and the primary antibody against Adamts5 (1:50) was acquired from HUABIO (Hangzhou, China).

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Staining

Journal: Aging (Albany NY)

Article Title: Lei’s formula attenuates osteoarthritis mediated by suppression of chondrocyte senescence via the mTOR axis: in vitro and in vivo experiments

doi: 10.18632/aging.205582

Figure Lengend Snippet: LSF ameliorates the progression of OA and inhibits mTOR expression in the mice DMM model. ( A ) Immunohisto- chemical typical images of pathological sections of mice knee joints from different experimental groups (SHAM, DMM, DMM+LSF-L (0.08 g/kg), DMM+LSF-M (0.17 g/kg), and DMM+LSF-H (0.35 g/kg)), which were examined for the detection of Col2, ACAN, Adamts5, MMP13, Col10, P16, P21 and mTOR expression in mice cartilage. (Scale bar. 50 μm). ( B – I ) Quantitative results of immunohistochemical staining in 5 groups of mice. All data represent mean ± SD. compared to the SHAM group; ****P < 0.0001; compared to the DMM group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies against CDKN2A/p16INK4a (1:100) were purchased from Zenbio (Chengdu, China), and the primary antibody against Adamts5 (1:50) was acquired from HUABIO (Hangzhou, China).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Flavokawain A alleviates the progression of mouse osteoarthritis: An in vitro and in vivo study

doi: 10.3389/fbioe.2022.1071776

Figure Lengend Snippet: Protective effects of FKA against IL-1β-induced inflammation and ECM destruction in mouse chondrocytes. Chondrocyte cell treatment with 5 ng/ml of IL-1β only or with FKA (20 and 40 μM) for a period of 24 h. Western blot analysis of inflammatory cytokines (COX2 and iNOS), catabolic (ADAMTS5, MMP3, and MMP13), and anabolic markers (Col2, Aggrecan, and Sox9) (A,C and E) . Quantitative analysis of protein expression (B,D and F) . (G and H) 5 ng/ml of IL-1β was added for treating the cells for 24 h in the absence or presence of 40 µM FKA. Confocal microscopy showing the expression of MMP13 and Aggrecan in immunofluorescence experiments (scale bar 10 μm). (I) Relative quantification of fluorescence intensity. The values are presented as means ± SD ( n = 3). # p < 0.05 vs the control group; * p < 0.05 and ** p < 0.01 vs the IL-1β group.

Article Snippet: ADAMTS5 primary antibody, secondary antibody, phosphate-buffered saline (PBS), trypsin, collagenase type II, the CCK8 assay kit, bovine serum albumin (BSA), and protein extraction kit were ordered and acquired from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Quantitative Proteomics, Fluorescence, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat

doi: 10.3389/fcell.2022.971736

Figure Lengend Snippet: Histological and immunofluorescence staining of intra-articular injection of rapamycin (A) . The result of HE staining (Scale bar: the left panel was 200 um, the right panel was 25 um) (B) . The result of Toluidine blue staining (Scale bar: the left panel was 200 um, the right panel was 25 um) (C) . The immunofluorescence results of Col2 (Scale bar: the left panel was 200 um, the right panel was 25 um) (D) . The immunofluorescence results of MMP13 (Scale bar: the left panel was 200um, the right panel was 25 um) (E) . HC/CC ratio under HE staining (F) . HC/CC ratio under toluidine blue staining (G) . The statistics of Col2 -positive cells (H) . The statistics of MMP13-positive cells. (I) . OARSI score in each group.

Article Snippet: HE staining solution, Toluidine blue staining powder and safranin O/fast green staining powder was buy from Sigma (United States), SDS-PAGE gel rapid preparation kit was attained from Beyotime Biotechnology (Shanghai)Co., Ltd. rapamycin was acquired from Shanghai yuanye Bio-Technology Co., Ltd., IL-1β was purchased from R&D Systems (Bio-Techne), 3-methyladenine (3-MA, autophagy inhibitor) was produced from TargetMol (United States), primary antibodies MMP13 (18165-1-AP), Adamts5 (Ag26730), P-AKT (Phospho-AKT1 (Ser473)), Beclin 1 (11306-1-AP), LC-3B (18725-1-AP), ULK1 (20986-1-AP), ATG12 (11264-1-AP), COL1 (66761-1-lg) achieved from Proteintech Group, COL2 (GB11021) were procured from Servicebio (China), P-S6K (9208S)/S6K (9202S) were come from Cell Signaling Technology (CST), P62 (ab109012) was purchased from Abcam (Shanghai).

Techniques: Immunofluorescence, Staining, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat

doi: 10.3389/fcell.2022.971736

Figure Lengend Snippet: The protective effect of rapamycin on the meniscus extracellular matrix. (A) The expression of MMP13, Col1, and Col2 in Western Blot (B) . The gray value statistics of MMP13 (C) . The gray value statistics of Col1 (D) . The gray value statistics of Col2 (E) . The result of safranin O/fast green staining (Scale bar: the left panel was 200 um, the right panel was 25 um) (F) . The immunofluorescence staining results of MMP13 (Scale bar: the left panel was 200 um, the right panel was 25 um) (G) . The statistics of MMP13-positive cells.

Article Snippet: HE staining solution, Toluidine blue staining powder and safranin O/fast green staining powder was buy from Sigma (United States), SDS-PAGE gel rapid preparation kit was attained from Beyotime Biotechnology (Shanghai)Co., Ltd. rapamycin was acquired from Shanghai yuanye Bio-Technology Co., Ltd., IL-1β was purchased from R&D Systems (Bio-Techne), 3-methyladenine (3-MA, autophagy inhibitor) was produced from TargetMol (United States), primary antibodies MMP13 (18165-1-AP), Adamts5 (Ag26730), P-AKT (Phospho-AKT1 (Ser473)), Beclin 1 (11306-1-AP), LC-3B (18725-1-AP), ULK1 (20986-1-AP), ATG12 (11264-1-AP), COL1 (66761-1-lg) achieved from Proteintech Group, COL2 (GB11021) were procured from Servicebio (China), P-S6K (9208S)/S6K (9202S) were come from Cell Signaling Technology (CST), P62 (ab109012) was purchased from Abcam (Shanghai).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat

doi: 10.3389/fcell.2022.971736

Figure Lengend Snippet: Influence of the meniscus on articular cartilage during the development of PTOA (A) . The immunofluorescence staining results of MMP13 in 1 week, 2 and 3 weeks after ACLT surgery (Scale bar: the left panel was 100 um, the middle and the right panel was 25 um). (B) The statistics of MMP13-positive cells in tibia (C) . The statistics of MMP13-positive cells in meniscus (D) . The expression of Adamts5 and MMP13 in Western Blot (E) . The gray value statistics of Adamts5 (F) The gray value statistics of MMP13.

Article Snippet: HE staining solution, Toluidine blue staining powder and safranin O/fast green staining powder was buy from Sigma (United States), SDS-PAGE gel rapid preparation kit was attained from Beyotime Biotechnology (Shanghai)Co., Ltd. rapamycin was acquired from Shanghai yuanye Bio-Technology Co., Ltd., IL-1β was purchased from R&D Systems (Bio-Techne), 3-methyladenine (3-MA, autophagy inhibitor) was produced from TargetMol (United States), primary antibodies MMP13 (18165-1-AP), Adamts5 (Ag26730), P-AKT (Phospho-AKT1 (Ser473)), Beclin 1 (11306-1-AP), LC-3B (18725-1-AP), ULK1 (20986-1-AP), ATG12 (11264-1-AP), COL1 (66761-1-lg) achieved from Proteintech Group, COL2 (GB11021) were procured from Servicebio (China), P-S6K (9208S)/S6K (9202S) were come from Cell Signaling Technology (CST), P62 (ab109012) was purchased from Abcam (Shanghai).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot