primary anti col ii  (Bio-Rad)

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: Protective effects of RUT against IL-1β-induced inflammation and extracellular matrix degradation in mouse chondrocytes. The cells were exposed to IL-1β (5 ng/ml) with/without RUT (1, 2.5, 5 and 10 μ M) for 24 h. (A and B) The protein expression levels of MMP3, MMP13, IL-1β, COX2, IL-6, TNF-α, SOX9, COL II and aggrecan were determined using western blot analysis. (C-G) Quantification analysis of the results of western blotting. (D and E) Relative mRNA expression levels of IL-6 and TNF-α in chondrocytes stimulated with IL-1β (5 ng/ml) and treated with or without RUT (1, 2.5, 5 and 10 μ M) for 24 h. The values are presented as the mean ± SD of three independent experiments. # P<0.05 vs. control group; ** P<0.01, *** P<0.001 and **** P<0.0001 vs. IL-1β group. (H) The protein expression of COL II was detected using immunofluorescence following treatment of the cells with IL-1β (5 ng/ml) with/without RUT (10 μ M) for 24 h. IL-1β, interleukin 1 β; RUT, rutaecarpine; MMP, matrix metalloproteinase; COX2, cyclooxygenase 2; SOX9, SRY-box transcription factor 9; COL II, collagen type II.

Article Snippet: Subsequently, primary antibodies against COL II (1:200; cat. no. 28459-1-AP, Proteintech Group, Inc.) and p65 (1:400; cat. no. 8242, Cell Signaling Technology, Inc.) were used to incubate the cells at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Rutaecarpine ameliorates osteoarthritis by inhibiting PI3K/AKT/NF-κB and MAPK signalling transduction through integrin αVβ3

doi: 10.3892/ijmm.2023.5300

Figure Lengend Snippet: The beneficial effects of RUT on IL-1β-stimulated chondrocytes were attenuated following integrin αVβ3 knockdown. (A and B) Relative mRNA levels of ItgαV and Itgβ3 in chondrocytes exposed to 5 ng/ml IL-1β with/without RUT (1, 2.5, 5 and 10 μ M) for 24 h. The knockdown efficiency of ItgαV and Itgβ3 siRNA transfection was verified using RT-qPCR. (C-E and H-M) The expression levels of inflammation and extracellular matrix degradation-related proteins were determined using western blot analysis; the outcomes of the chondrocytes were quantified in the presence/absence of 5 ng/ml IL-1β, RUT (10 μ M) and ItgαVβ3 for 24 h. (F and G, and N-S) Western blot analysis and quantification analysis were used to measure the levels of PI3K/Akt/NF-κB and MAPK-related signalling proteins in IL-1β (5 ng/ml)-stimulated chondrocytes for 30 min following transfection with/without ItgαVβ3 siRNA and RUT (10 μ M) for 24 h. Data are presented as the mean ± SD of three independent experiments. # P<0.05 vs. control group; * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 vs. IL-1β group; ns, no significant difference (P>0.05). IL-1β, interleukin 1 β; RUT, rutaecarpine; MMP, matrix metalloproteinase; COX2, cyclooxygenase 2; SOX9, SRY-box transcription factor 9; PI3K, phosphoinositide-3-kinase; COL II, collagen type II; AGG, aggrecan.

Article Snippet: Subsequently, primary antibodies against COL II (1:200; cat. no. 28459-1-AP, Proteintech Group, Inc.) and p65 (1:400; cat. no. 8242, Cell Signaling Technology, Inc.) were used to incubate the cells at 4°C overnight.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Control