Journal: PLoS ONE

Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: (A) Five μg of human lubricin purified from synovial fluid (PHL), and 1 μl of synovial fluid recovered from patients with osteoarthritis (OA), CACP, or from bovine, porcine, goat, dog, rat and guinea pig were electrophoresed under reducing conditions on 4–20% SDS-PAGE gradient gels, transferred to PVDF, and immunodetected using the biotin labeled mAbs. Note that the mAbs detect protein in the synovial fluid from patients with OA and from other species, but not from the patient with CACP, who is genetically deficient for lubricin. (B) 0.7 μg of Purified human lubricin (PHL), porcine gastric mucin (PM) and bovine submaxillary mucin (BSM) were electrophoresed under non-reducing conditions on 4–12% SDS-PAGE gradient gels, transferred to nitrocellulose and immunodetected using the mAbs. For mAb-8e3, 1.4 μg of PHL was loaded. Note that anti-lubricin mAbs did not cross react with BSM or PM.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Purification, SDS Page, Labeling

Journal: PLoS ONE

Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Expressing, Recombinant, SDS Page, Modification, Mutagenesis, Variant Assay, Labeling

Journal: PLoS ONE

Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, Concentration Assay, Recombinant

Journal: PLoS ONE

Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Labeling, Purification, SDS Page, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Immunodetection

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: Loss of Prmt5 in osteochondral progenitor cells induces early-onset scoliosis in mice. (A) Col2Cre;Prmt5 f/f mutant mice were produced in Mendelian ratios at E18.5 (22.2%, n =18), but displayed progressive mortality prior to weaning (11.9%, n =42 at P1; 5.4%, n =62 at P10; 0%, n =23 at P16). (B,C) Skeletal preparations showed comparable size and normal patterning of the spine in Col2Cre;Prmt5 f/f mutant mice at E18.5 compared with the littermate Cre – controls ( n =4 for each group). (D-F″) X-ray imaging analysis and quantification at P10 demonstrating early-onset thoracic scoliosis (F,F′, red arrowheads) and increased Cobb angle in mutant mice (D) ( n =4 for each group; each dot represents one mouse analyzed, plotting with mean± s.d.; ** P <0.01, two-tailed Student’s t -test). Sagittal X-ray image showing reduced ossification of the distal ribs in the mutant (F″) compared with a littermate control (E″, yellow arrowheads). (G-J) MicroCT scanning of the thoracic region of the spine (T5-T7) shows smaller vertebrae and a mildly wedged vertebral body at the curvature (T6) in mutant mice (H) compared with the controls (G) at P10. Sagittal sections of the microCT three-dimensional reconstruction of the thoracic vertebral bodies shows reduced bone formation within the vertebral body in the mutant mice (J) compared with controls (I) at P10. (K-L′) Immunohistochemical (IHC) analysis of PRMT5 in thoracic spine sections of control (K) and mutant (L) mice at P10. The boxes outline the areas shown at higher magnification in K′ and L′. Scale bars: 2 mm (B,C); 10 mm (E,F); 50 mm (E′,E″,F′,F″); 1 mm (G-J); 100 µm (K-L′). AF, annulus fibrosus; EP, endplate; GP, growth plate; NP, nucleus pulposus.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Mutagenesis, Produced, Imaging, Two Tailed Test, Immunohistochemical staining

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: Loss of Prmt5 in osteochondral progenitor lineages of the spine results in defective ossification of the vertebral body. (A-D) Alcian Blue Hematoxylin/Orange G (ABH/OG) staining of thoracic spines of Cre – control (A,C) or Col2Cre;Prmt5 f/f mutant (B,D) mice at P1 (A,B) and P10 (C,D). The boxes outline the areas shown at higher magnification in A′, A″, B′, B″, C′, C″, D′ and D″. (A″,B″) P1 adjacent sections stained with Safranin O/Fast Green (SO/FG). (B) Impaired ossification in Col2Cre;Prmt5 f/f mutant vertebrae (yellow asterisk). (B′,B″) Representative images of midline clefts consistently observed in mutant endplate, but less common in Cre – control mice (A′,A″) (yellow arrowheads; n =6 for controls and n =5 for mutants). (D) At P10, regions of asymmetric defects of bone formation observed in the vertebrae of Col2Cre;Prmt5 f/f mutant mice (D, yellow asterisks), which are ossified in Cre – control mice (C). The organization of chondrocytes in regions of persistent cartilage were found in columns (D″, red arrow/dashed outline) and clusters (D″, yellow arrow/dashed outline) ( n =3 for each group). Endplate, proliferative/prehypertrophic growth plate and hypertrophic growth plate are labeled with green, orange and red brackets, respectively. The regions of the thoracic spine checked (T8, T9) are labeled on the vertebrae. (E) Quantification of the percentile of IVDs contain midline clefts in control and mutant IVD at P1 as shown in B-B″. Each dot represents one mouse analyzed, plotting with mean±s.d. ( n =6 mice for controls and n =5 mice for mutants). Four to six thoracic IVDs were checked for each mouse. (F) Quantification of the number of cell layers of the endplate and growth plate in control and mutant mice at P10 as shown in C′ and D′. Each dot in the box and whisker plot represents the counts from a single IVD and adjacent growth plate analyzed. Each IVD and growth plate was analyzed three times and averaged, and at least two IVDs and growth plates were analyzed per mouse ( n =3 mice for each group). * P <0.05, two-tailed Student's t -test. Scale bars: 100 µm.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Staining, Mutagenesis, Labeling, Whisker Assay, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: Loss of Prmt5 in osteochondral progenitors results in induced expression of COLX and depleted expression of PRG4 in the intervertebral disc. (A-D′) IHC analysis of type X collagen (COLX) (A,B) and proteoglycan 4 (PRG4) (C,D) in thoracic spine sections of Cre – control (A,C) or Col2Cre;Prmt5 f/f mutant (B,D) mice at P10. The boxes outline the areas shown at higher magnification in A′-D′. Mutant mice demonstrated increased COLX signal in the endplate (B′, red arrowheads) and growth plate, and diminished PRG4 signal in the endplate and growth plate (D′), which was consistently observed in the Cre – control mice (C′, red arrowheads) ( n =3 mice for each group). Scale bars: 100 µm. AF, annulus fibrosus; EP, endplate; GP, growth plate; hGP, hypertrophic growth plate; NP, nucleus pulposus; pGP, proliferative growth plate.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Expressing, Mutagenesis

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: PRMT5 regulates the expression of RUNX2, Mmp13 and Ihh . (A,B) IHC analysis of RUNX2 in thoracic spine sections of Cre – control (A) or Col2Cre;Prmt5 f/f mutant (B) mice at P10, demonstrating reduced RUNX2 expression in the endplate and growth plate of the mutant mice. The boxes outline the areas shown at higher magnification in A′ and B′. (C,D) Fluorescent in situ hybridization (FISH) analysis of Mmp13 in thoracic spine sections of Cre – control (C) or Col2Cre;Prmt5 f/f mutant (D) mice at P10, with 4′,6-diamidino-2-phenylindole (DAPI; nuclei) in blue. Reduced Mmp13 signal was detected in the growth plate of the mutant mice. C′ and D′ are grayscale Mmp13 fluorescent in situ channels; C, C″, D and D″ are pseudocolored merged channels. The boxes outline the areas shown at higher magnification in C′, C″, D′ and D″. (E,F) FISH analysis of Ihh in thoracic spine sections of Cre – control (E) or Col2Cre;Prmt5 f/f mutant (F) mice at P10. Induced expression of Ihh was detected in the growth plate of the mutant mice. E′ and F′ are grayscale Ihh fluorescent in situ channels; E, E″, F and F″ are pseudocolored merged channels. The boxes outline the areas shown at higher magnification in E′, E″, F′ and F″ ( n =3 for each group). Scale bars: 100 µm. AF, annulus fibrosus; EP, endplate; GP, growth plate; NP, nucleus purposes.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Expressing, Mutagenesis, In Situ Hybridization, In Situ

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: Postnatal ablation of Prmt5 in cartilaginous tissues of the spine resulted in alterations in normal gene expression. (A) Schematic of the protocol for postnatal recombination of the floxed Prmt5 allele used for these experiments. (B,C) Beta-galactosidase staining in thoracic spine sections of Cre – control (B) or ATC;Rosa-LacZ f/+ (C) mice at 5 weeks of age shows that effective recombination is achieved within the IVD and growth plate at 1 week post-Dox induction. The boxes outline the areas shown at higher magnification in B′ and C′. (D) Real-time RT-PCR analysis of IVD and growth plate tissues harvested at 2.5 months of age demonstrates effective reduction in Prmt5 as well as Mmp13 and Prg4 . Bars represent mean±s.d. ( n =3 for each group). * P <0.05, ** P <0.01, two-tailed Student's t -test. (E,F) Safranin O/Fast Green staining in thoracic spine sections of Cre – control (E) or ATC;Prmt5 f/f mutant (F) mice harvested at 4 months of age after Dox treatment. No obvious histopathology was observed except for the occurrence of minor clefts of the endplate and growth plate in the mutant mice (F, yellow arrow). (G,H) IHC analysis of COLX protein in thoracic spine sections from Cre – control (G) or ATC;Prmt5 f/f mutant (H) mice at 4 months of age. The boxes outline the areas shown at higher magnification in G′ and H′. Increased and ectopic COLX signal was observed in the endplate (H′, black arrows) and growth plate (H′, red arrows) of the mutant mice ( n =3 for each group). Scale bars: 100 µm. AF, annulus fibrosus; EP, endplate; GP, growth plate; NP, nucleus pulposus.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Expressing, Staining, Quantitative RT-PCR, Two Tailed Test, Mutagenesis, Histopathology

Journal: Disease Models & Mechanisms

Article Title: Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis

doi: 10.1242/dmm.041251

Figure Lengend Snippet: A model of PRMT5 in regulating terminal differentiation of hypertrophic chondrocytes in the perinatal vertebral growth plate of the spine. (A) PRMT5 expressed in proliferative and prehypertrophic chondrocytes functions to regulate chondrocyte hypertrophic differentiation by controlling RUNX2 and IHH signaling. PRMT5 is required for maintaining postnatal expression of RUNX2, which is a key regulator of terminal differentiation of hypertrophic chondrocytes, and a positive regulator of MMP13 expression. PRMT5 also negatively regulates the expression of Ihh . (B) Prmt5 deletion in cartilaginous tissues of the spine leads to expanded Ihh expression in the perinatal growth plate and promotes the expression/accumulation of COLX. It also results in reduced RUNX2 and Mmp13 expression, which inhibits the terminal differentiation of the hypertrophic chondrocytes. These two processes synergistically lead to persistent hypertrophic growth plate and reduced bone formation in perinatal mouse vertebrae. AF, annulus fibrosus; EP, endplate; IVD, intervertebral disc; NP, nucleus pulposus; WT, wild type.

Article Snippet: Colorimetric development was performed with the following primary antibodies: anti-PRMT5 (Abcam, ab109451, 1:100), anti-COLII (Thermo Fisher Scientific, MS235B, 1:100), anti-COLX (Quartett, 1-CO097-05, 1:200), anti-SOX9 (Santa Cruz Biotechnology, sc-20095, 1:50), anti-Lubricin (PRG4) (Abcam, ab28484, 1:400) and anti-RUNX2 (Medical & Biological Laboratories, D130-3, 1:100).

Techniques: Expressing

Journal: bioRxiv

Article Title: Overexpression of Mig-6 in Limb Mesenchyme Leads to Accelerated Osteoarthritis in Mice

doi: 10.1101/871350

Figure Lengend Snippet: Representative SOX9 immunostaining in knee joints of 12 week-old male control (A) or Mig-6 overexpressing (B) mice (n=5 mice/group). Overexpressing mice showed reduced numbers of positive cells in the medial and lateral compartments. LFC = lateral femoral condyle, LTP = lateral tibial plateau, MFC = medial femoral condyle and MTP = medial tibial plateau. Scale bar = 100μm.

Article Snippet: Primary antibodies against SOX9 (R&D Systems, AF3075), MMP13 (Protein Tech, Chicago, IL, USA, 18165-1-AP), and lubricin (Abcam, ab28484) were used and slides without primary antibody were used as control.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Overexpression of Mig-6 in Limb Mesenchyme Leads to Accelerated Osteoarthritis in Mice

doi: 10.1101/871350

Figure Lengend Snippet: Representative SOX9 immunostaining in knee joints of 36 week-old male control (A) or Mig-6 overexpressing (B) mice (n=5 mice/group). Overexpressing mice showed reduced numbers of positive cells in the medial and lateral compartments. LFC = lateral femoral condyle, LTP = lateral tibial plateau, MFC = medial femoral condyle and MTP = medial tibial plateau. Scale bar = 100μm.

Article Snippet: Primary antibodies against SOX9 (R&D Systems, AF3075), MMP13 (Protein Tech, Chicago, IL, USA, 18165-1-AP), and lubricin (Abcam, ab28484) were used and slides without primary antibody were used as control.

Techniques: Immunostaining