Journal: Osteoarthritis and cartilage

Article Title: An ARGS-aggrecan assay for analysis in blood and synovial fluid.

doi: 10.1016/j.joca.2013.12.010

Figure Lengend Snippet: Fig. 1. Aggrecan fragments in cartilage, SF and blood detected by Western blot. (A) Large aggrecan fragments in cartilage are degraded into smaller fragments seen in SF, and this process (arrows) continues in SF and blood. Aggrecan fragments were purified by: A1D1 preparation from a knee cartilage OA-pool, D1 preparations from an SF acute injury pool, and serum and plasma pools. Purified aggrecan samples were deglycosylated and analysed (0.8e6 mg GAG per lane, 3e8% Triseacetate gels) by anti-ARGS, -FFGV and -G1 Western blot. The Western blot immunosignal was blocked by immunisation peptides (not shown). Representative Western blot images from full sized blotted gels with aggrecan (molecular weights in kDa) are shown. P, plasma. S, serum. (B) Captured and extracted aggrecan fragments from the wells of MSD plates coated with AHP0022 anti-G1/G2 antibody. Material from 96 wells of each samples: SF, serum (S), ADAMTS-4-digested A1D1 aggrecan (A þ TS4), and dilution buffer (BU) as negative control e were extracted, pooled and analysed on the anti-ARGS Western blot. The A þ TS4 sample was also run directly on the gel (0.75 mg GAG) as a positive control. False-positive immunobands are indicated by asterisks (*).

Article Snippet: As ARGS standard, we used recombinant human aggrecan G1G2 (20VETS-CFRG675, 1220-PG, R&D Systems) digested with ADAMTS-4 for complete conversion to G1-TEGE and ARGS-G2 fragments.

Techniques: Western Blot, Clinical Proteomics, Negative Control, Positive Control

Journal: Osteoarthritis and cartilage

Article Title: An ARGS-aggrecan assay for analysis in blood and synovial fluid.

doi: 10.1016/j.joca.2013.12.010

Figure Lengend Snippet: Fig. 2. The human recombinant G1-G2 peptide is completely degraded by aggreca- nase. Recombinant human aggrecan G1-G2 peptide (20VETS-CFRG675, R&D #1220-PG) was digested by ADAMTS-4 for 24 h at 37C. (A) Samples (0.18e0.31 mg/well) were run on a 4e12% Bis-Tris SDS-PAGE gel as a digest (þ) or as a G1-G2 peptide (), and aggrecan fragments were visualised by silver staining29 or by Western blot using G1/ G2 (AHP0022), G1, TEGE and ARGS-aggrecan antibodies. Representative silver stain and Western blot images from full sized blotted gels with aggrecan (molecular masses in kDa) are shown. *, silver stained false-positive none G1-G2 peptide bands below 37 kDa are marked. (B) Illustration of aggrecan G1-TEGE and ARGS-G2 fragments and the recognition by the anti-G1/G2 and -ARGS antibodies. In the ARGS ELCL assay, anti- G1/G2 is used as the capture antibody and anti-ARGS as the detection antibody.

Article Snippet: As ARGS standard, we used recombinant human aggrecan G1G2 (20VETS-CFRG675, 1220-PG, R&D Systems) digested with ADAMTS-4 for complete conversion to G1-TEGE and ARGS-G2 fragments.

Techniques: Recombinant, SDS Page, Western Blot, Silver Staining, Staining

Journal: Journal of Biological Chemistry

Article Title: PP2A Regulates BCL-2 Phosphorylation and Proteasome-mediated Degradation at the Endoplasmic Reticulum

doi: 10.1074/jbc.m602648200

Figure Lengend Snippet: FIGURE 4. Knock down of PP2A regulatory B subunit B56 affects BCL-2 protein levels. A, small hairpin RNA knock down of B56 (shB56) in immor- talized HEK cells. To assess knock down, B56 was immunoprecipitated from 1 mg of lysate using a mouse monoclonal antibody, and a Western blot for B56 was done with a rabbit polyclonal antibody (21). Protein levels of BCL-2, BAX, and actin were assessed from whole cell lysate. B and C, control and shB56 cells were incubated with and without MG132 for 8 h. BCL-2 and BCL-XL protein levels were assessed by Western blot from whole cell lysate. B, B56-overexpressing cells were also incubated with MG132. C, B56 overex- pression levels were assessed from whole cell lysate using the mouse mono- clonal antibody.

Article Snippet: To immunoprecipitate B56 , mouse monoclonal antibodies were generated from full-length B56 3 (distributed by Novus or Zymed Laboratories Inc.).

Techniques: Knockdown, Immunoprecipitation, Western Blot, Control, Incubation

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown by small interfering RNA (siRNA) on chondrocyte phenotype. A The human chondrocyte cell line C20A4 was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and MMP-13 protein expression was analyzed by Western blotting. B Chondrocyte-related gene expression, including SOX9, aggrecan (ACAN), MMP13, type X collagen (COL10A1), and type II collagen (COL2A1), was assessed by quantitative PCR in siEFEMP1- and siCtrl-treated C20A4 cells. C Phosphokinase protein arrays were used to profile phosphorylation changes in intracellular proteins extracted from C20A4 chondrocytes. D Spot intensities exhibiting changes greater than ± 25% between groups were quantified using ImageJ software. Red lines indicate baseline levels from the siCtrl group. E Phosphorylation of p70S6K at threonine 421/serine 424 (T421/T424) was further examined by Western blot analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using an independent-samples t-test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Small Interfering RNA, Transfection, Expressing, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Immunofluorescence analysis of cartilage markers following EFEMP1 antibody treatment in STR/ort mice. A Representative immunofluorescence (IF) staining for EFEMP1, SOX9, MMP13, aggrecan fragments, COL2A1, and COL10A1 in the medial femoral condyle (MF) and medial tibial plateau (MT). Nuclei were counterstained with DAPI (blue), and articular cartilage regions are outlined by orange dashed lines. B Quantification of fluorescence intensity in articular cartilage regions of the femoral condyle and tibial plateau, with medial and lateral compartments combined. EFEMP1 and SOX9 signals were normalized to DAPI, whereas MMP13, aggrecan fragments, COL10A1, and COL2A1 signals were quantified as absolute FITC intensity. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using an independent-samples t -test. P < 0.05, P < 0.01, and ** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Immunofluorescence, Staining, Fluorescence