Anti-Collagen II/COL2A1 Antibody Picoband®

Anti-Collagen II antibody, PA2141-1, IHC(F)
IHC(F): Rat Trachea Tissue

Anti-Collagen II antibody, PA2141-1, Western blotting
Lane 1: Rat Heart Tissue Lysate
Lane 1: Rat Brain Tissue Lysate

SESN2 overexpression improves fatty acid metabolism disorders and ameliorates cartilage degeneration (A and B) IF (A) and corresponding quantitative analysis (B) of SESN2 in the articular cartilage of mice induced by sham or destabilization of medial meniscus (DMM) surgery with intraarticular injection of lv-nc and lv-Sesn2 (n = 6). (C–E) S.O. staining (C) of mice treated as in (A) (n = 6). Quantitation of Osteoarthritis Research Society International (OARSI) scores (D), chondrocyte numbers, and cartilage thickness (E) (n = 6). (F and G) IHC (F) and corresponding quantitative analysis (G) of COL2A1 and MMP13 in the articular cartilage of mice treated as in (A) (n = 6). (H and I) BODIPY493/503 staining, IF (H) of SREBP1, FASN, and SCD1, and corresponding quantitative analysis (I) in the articular cartilage of mice treated as in (A) (n = 6). (J and K) IF of PGC-1α and TUNEL staining (J) and corresponding quantitative analysis (K) in the articular cartilage of mice treated as in (A) (n = 6). Scale bars: (F, H, and J) 50 μm, (C) 50 and 100 μm. Data (n = 6) are shown as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Statistical analysis was performed by one-way ANOVA (B, D, E, G, I, and K).
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DMOG enhances the therapeutic potential of senescent MSCs to mitigate metabolic dysfunction in OA chondrocytes. ( A ) Schematic diagram of the co-culture system. Human articular chondrocytes were treated with IL-1β to induce OA-like conditions and co-cultured with MSCs from various experimental groups using a Transwell system. ( B ) Representative immunofluorescence staining of ECM-related proteins, including Collagen II (Col2a1), Aggrecan (AGC), and MMP13, in OA chondrocytes co-cultured with MSCs from different experimental groups. Scale bar = 50 μm. ( C ) Quantitative analysis of immunofluorescence intensity for Col2a1, AGC, and MMP13. ( D ) Quantitative real-time PCR (qRT-PCR) analysis of mRNA levels for Col2a1, AGC, and MMP13 in OA chondrocytes co-cultured with MSCs. Data are expressed as mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 Full size image
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Down-regulation of Notch1 signaling promoted BMP2-induced chondrogenic differentiation and inhibited BMP2-induced osteogenic differentiation of MSCs in vitro. (A) C3H10T1/2 cells were transfected with AdGFP, AdBMP2, AdDnNotch1, and AdBMP2+DnNotch1, respectively. On day 7, quantitative reverse transcription PCR was used to detect the expression of chondrogenic differentiation markers (Sox9 and Col2a1) and osteoblastic differentiation markers (Runx2, Col1a1, and OPN) of MSCs. (B) To detect the expression of sulfated glycosaminoglycan during C3H10T1/2 cell differentiation, alcian blue staining was performed on day 7 after cells were transfected with recombinant adenovirus. (C) Alkaline phosphatase (ALP) staining experiments were used to determine ALP activity on day 3 (a) and day 7 (b) respectively. (D) For matrix mineralization, alizarin red S staining was performed on day 14 (a); microscopic (b) observations showed that down-regulation of Notch1 signaling inhibited BMP2-induced calcium deposition. (E) Quantitative analysis of ALP activities and calcium deposition. The ALP activity was quantified at OD 405 nm and normalized by protein concentration per well (unit/mg protein) on day 3 (a) and day 7 (b). Alizarin red staining was quantified at OD 405 nm and normalized to total DNA per well (OD405 nm/μg DNA) (c). (F) Western blot analysis for the chondrogenic differentiation markers Sox9 and Col2a1 and the osteogenic markers Runx2, Col1a1, and OPN. Protein bands (a) and quantitative analysis (b). The relative expression of Sox9, Col2a1, Runx2, Col1a1, and OPN proteins were analyzed using GAPDH as control (b). One-way analysis of variance; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 versus the AdGFP group; ####p < 0.0001, ###p < 0.001, ##p < 0.01, and #p < 0.05 versus the indicated group; ns, p > 0.05. BMP2, bone morphogenetic protein 2; Col1a1, collagen type I alpha 1 chain; Col2a1, collagen type II alpha 1 chain; MSC, mesenchymal stem cell; Notch1, Notch receptor 1; OPN, osteopontin; Runx2, RUNX family transcription factor 2; Sox9; SRY-box transcription factor 9.
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MSMP affected cartilage matrix metabolism in OA mice. (A) Immunohistochemical staining of Col2a1. (B) Statistical analysis of the positive expression for Col2a1. (C) Immunohistochemical staining of Mmp13. (D) Statistical analysis of the positive expression for Mmp13. Scale bar represents 100 μm (Date are mean ± SD, #### p < 0.0001, versus Sham group; * p < 0.05, ** p < 0.01, **** p < 0.0001, versus OA group).
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Immunohistochemical staining of COL2A1 in vivo at indicated times postoperation. Scale bar: 200 μm
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Hoxc10 is positively correlated with cartilage. n (C) q‐PCR validated the expression levels of Sox9 gene after overexpression and knockout of Hoxc10. (D) q‐PCR validated the expression levels of the Col2a1 gene after overexpression and knockout of Hoxc10. (E) q‐PCR validated the expression levels of Aggrecan gene after overexpression and knockout of Hoxc10. (F) The proteoglycan of BMSCs after overexpression and knockdown Hoxc10 was observed by Alcian blue staining 21 days after chondrogenic induction. (G) Quantitative analysis of Alcian blue staining. (H) Schematic diagram of co‐culture of L‐BMSCs and M‐BMSCs with and without Hoxc10 knockout. (I) Col2a1 gene expression in L‐BMSCs after Hoxc10 knockout and co‐culture with M‐BMSCs compared to control. (J) ChIP experiment of Sox9 and Hoxc10 protein binding. The data are presented as the mean ± SD ( n = 3).* p < 0.05.
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Hoxc10 is retained in L/M‐BMSCs in vitro. (A) Schematic of Transwell co‐culture model of L‐BMSCs and M‐BMSCs. (B) Schematic of the limb bones and mandibles from different embryonic origins. The mandible is of neural crest origin (blue) and the limb bone is of mesodermal origin (orange) (C) qPCR verified the gene expression levels of Sox9 and Col2a1 before and after co‐culture of L‐BMSCs and M‐BMSCs. (D) The proliferation, osteogenic and chondrogenic genes of femoral homotopic grafting, femoral heterotopic grafting and mandibles homotopic grafting. (E) After 21 days of chondrogenic induction in the upper layer cells of Transwell model before and after co cultivation with L‐BMSCs and M‐BMSCs, blue stained proteoglycans were observed using Alcian blue. (F) Quantitative analysis of Alizarin blue staining before and after co culture of L‐BMSCs and M‐BMSCs. The data are presented as the mean ± SD ( n = 3). * p < 0.05.
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SiRNA TLR7 reversed collagen II and ADAMTS-5 degradation in IL-1β-stimulated chondrocytes. Cells were stimulated with IL-1β (10 ng/ml) for 48 h. The expression levels of collagen II and ADAMTS-5 were evaluated by Western blot ( a ) and quantification analysis ( b ). *** P < 0.001 versus the control group, ### P < 0.001 versus the IL-1β group.
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Magnetothermal suppression of macrophagic inflammation and chondrocyte ferroptosis by MNPs-TRPV1. (A) Cell viability of RAW264.7 macrophages treated with different concentrations of MNPs-TRPV1 with (w/) or without (w/o) AMF stimulation. (B) The inductively coupled plasma mass spectrometry (ICP-MS) detection of iron content per RAW264.7 cell after incubated with 5 mg/ml MNPs or MNPs-TRPV1 for 3, 6, and 12 h. (C and D) Flow cytometry analysis (C) and quantitative analysis (D) of sulfo-cyanine3 (Cy3) conjugated MNPs or MNPs-TRPV1 incubated RAW264.7 cells for 12 h. The representative image presented in (C) showed the combination of MNPs-TRPV1 to the plasma membrane of RAW264.7 cell. (E) Western blot analysis of the expression levels of CaMKII and p-CaMKII in RAW264.7 cells 0, 5, 10, and 15 min after the treatment of MNPs-TRPV1 with AMF stimulation. (F) qPCR analysis of inflammatory genes, including Il-1β, Il-6, Tnf-α, Il-18, and Ptgs2, in RAW264.7 cells treated with or without 50 ng ml−1 LPS in the presence or absence of MNP-TRPV1 pretreatment under AMF exposure. (G) Western blot analysis (left panel) and corresponding quantitative analysis (right panel) of the expression of inflammatory proteins, iNos and Cox2, expressed in RAW264.7 cells induced as indicated. (H) Immunofluorescence staining (left panel) and quantitative analysis (right panel) of iNos and Cox2 in RAW264.7 cell induced as indicated. (I) Cell viability of mouse primary chondrocytes treated with various concentrations of MNPs-TRPV1 with (w/) or without (w/o) AMF stimulation. (J) The ICP-MS detection of iron content per chondrocyte after incubated with 5 mg/ml MNPs or MNPs-TRPV1 for 3, 6, and 12 h. (K and L) Flow cytometry analysis (C) and quantification (D) of Cy3-conjugated MNPs or MNPs-TRPV1 incubated chondrocytes for 12 h. The representative image presented in (K) showed the combination of MNPs-TRPV1 to the plasma membrane of chondrocyte. (M) The proteins expression levels of CaMKII and p-CaMKII in chondrocytes treated as indicated at the time point of 0, 5, 10, and 15 min. (N) qPCR analysis of gene expression levels of ferroptosis suppressors (Gpx4, Fth1, Slc7a11, and Cd44), ferroptosis drivers (Ptgs2, Ncoa4, Cdo1, Atf3, Pgd, and Tfrc), cartilage anabolic marker (Col2a1), and cartilage catabolic marker (Mmp13) in chondrocytes treated as indicated. (O and P) Representative images (left panel) and quantification (right panel) of ROS (O) and lipid ROS (P) levels in mouse primary chondrocytes treated with TBHP (T) or T + MNPs-TRPV1 under the stimulation of AMF. (Q) Western blot analysis of expression levels of Col II, Mmp13, Fth1, and Gpx4 in chondrocytes treated as indicated. w/o, without; w/, with. Scale bars, 15 μm (C and K), 20 μm (H) and 50 μm (O and P). One-way (D, F, G, H, and L) or 2-way (A, B, I, J, O, and P) ANOVA with Tukey’s post hoc test. Data are shown as mean ± SD.
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Fap degrades denatured or MMP13-cleaved Col II. a Fap degrades denatured Col II in a dose-dependent manner. Denatured Col II (boiled at 95 °C for 10 min) was incubated with different amounts of rFap at 37 °C for 24 h. Samples were separated by SDS‒PAGE and quantified by colloidal blue staining ( n = 3 independent experiments). b Fap degrades denatured Col II in a time-dependent manner. Denatured Col II was incubated with rFap at 37 °C for 0–24 h ( n = 3 independent experiments). c FAPi inhibits Fap-mediated degradation of denatured Col II. Different doses of FAPi were preincubated with rFap for 30 min before denatured Col II was added and further incubated at 37 °C for 24 h ( n = 3 independent experiments). d Immunoprecipitation of Fap. HEK293T cells were transfected with GFP control, Oln-Flag, Fap-HA, or Oln-Flag + Fap-HA. Two days after transfection, Fap was immunoprecipitated from total cell lysates with 10 μL anti-HA affinity gel. Five percent total cell lysates were loaded as an input control ( n = 3 independent experiments). e Oln inhibits the Fap-mediated degradation of denatured Col II. Col II was coincubated with immunoprecipitated samples (in 10 μL anti-HA affinity gel) at 37 °C for 24 h ( n = 3 independent experiments). f Fap degrades MMP13-cleaved Col II. Native Col II was preincubated with rFap or rMMP13 at 37 °C for 12 h. EDTA was then added to the reaction mixture with or without rFap and incubated at 37 °C for another 12 h ( n = 3 independent experiments). g Grayscale quantification of the 55, 40 and 30 kDa digestion bands in ( f ). h Experimental design. Primary chondrocytes were stimulated with vehicle control (PBS), 200 ng·mL −1 rFap, 200 ng·mL −1 rMMP13, or 200 ng·mL −1 rFap plus 200 ng·mL −1 rMMP13 for 24 h. i Western blot analysis of mouse Col2a1 protein levels in primary chondrocytes stimulated as in ( h ) ( n = 3 independent experiments). The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001)
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UPR-related gene/protein expression in four groups. a Quantitative RT-PCR for ATF6 , ATF4 , and XBP1 of the four groups in the cultured constructs on 0, 7, 14, and 21 days. These data were normalized to GAPDH . b Western blot results for ATF4, ATF6, XBP1, and COL2A1 at different culture times. COL2A1 was not detectable at 0 day; β-actin was used as a loading control. c Normalized expression of ATF4, ATF6, XBP1, and COL2A1 in response to Western blot analysis. d Immunofluorescence staining of ATF4, ATF6, and XBP1 at 0 days. Scale bar: 50 μm. The values were means ± S.D.; * indicate P < 0.05, ** indicate P < 0.01, *** indicate P < 0.001
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Chondrogenic-related gene/protein expression in four groups. a Quantitative RT-PCR for ACAN , SOX9 , COL2A1 , and COL1A1 of the four groups in the cultured constructs on 7, 14, and 21 days. These data were normalized to GAPDH . b Immunofluorescence staining of COL2A1 after 21 days of chondrogenic induction. Scale bar: 25 μm. c The semi-quantitative analysis of positive staining of COL2A1 in response to Fig. b. d MTT assay was used to analyze cell proliferation of the four groups: P0 BMSCs, P0 + 4-PBA, P3 BMSCs and P3 + TM. e The DNA and GAG contents were stained for the Hoechst 33258 and dimethylmethylene blue dye binding assays, respectively, after 7, 14 and 21 days of chondrogenic induction, and the absorbances were measured to quantify the contents of DNA and GAG. The left panel shows the DNA content and the right panel shows the ratio between GAG and DNA content. f GAG production was examined using Safranin-O staining in the four samples: P0 BMSCs, P0 with 4-PBA treatment, P3 BMSCs and P3 with TM treatment at 21 days. Scale bar: 100 μm. The values are means ± S.D.; * indicate P < 0.05, ** indicate P < 0.01, *** indicate P < 0.001
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IF analysis of COL2A1 using anti-COL2A1 antibody (PA2141-1).
COL2A1 was detected in a paraffin-embedded section of Mouse cartilage tissue tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:100 rabbit anti-COL2A1 Antibody (PA2141-1) overnight at 4°C. Donkey anti-rabbit Alexa Fluor 555was used as secondary antibody at 1:500 dilution and incubated for 1 hour at RT. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.