Aggrecan from bovine articular cartilage

Aggrecan from bovine articular cartilage (cat no. A1960), and poly-L-ornithine hydrobromide (Mw 30,000–70,000; cat no. P3655) were purchased from Sigma-Aldrich. Recombinant human pleiotrophin produced in yeast was described previously [37 (link), 41 (link), 60 (link), 61 (link)]. Anti-PTPRZ-S, rabbit polyclonal antibodies against the extracellular region of PTPRZ [62 (link)], and rabbit polyclonal antibodies against phosphorylated Tyr-1105 of p190 RhoGAP [43 (link)] were described previously. The following are the sources of commercially available regents and antibodies used in the present study: Anti-aggrecan (clone Cat-315; cat no. MAB1581, Millipore), anti-MBP (cat no. sc-13914, Santa Cruz Biotechnology), anti-NG2 proteoglycan (cat no. AB5320, Millipore), anti-phosphotyrosine (clone PY20; cat no. ab16389, Abcam), anti-p190 RhoGAP (cat no. 610150, BD Biosciences; and cat no. 12164, Cell Signaling Technology), anti-FYN (cat no. P2992, Sigma-Aldrich; and cat no. 4023, Cell Signaling Technology), anti-phosphorylated Tyr-416 of Src (cat no. 2101, Cell Signaling Technology), anti-phosphorylated Tyr-527 of Src (cat no. 2105, Cell Signaling Technology), anti-GFAP (cat no. Z0334, Dako), anti-OLIG2 (cat no. AB9610, Millipore), and anti-APP (cat no. ab15272, Abcam).

We performed serum autoantibody ELISA with selected autoantigens to validate the microarray findings. Ninety-six-well MaxiSorp ELISA plates (Nunclon, Thermo Fisher Scientific, Waltham, MA, USA) were coated with 2 ug/ml of the following antigens in PBS: complement C1q (Biodesign), histone H1, histones H2A and H4, histone H2B (Immunovision, Springdale, AR, USA), collagen types IV and X from human placenta (Sigma-Aldrich, St Louis, MO, USA), plasmid dsDNA (Diarect, Freiburg, Germany), aggrecan from bovine articular cartilage (Sigma-Aldrich), or bovine serum albumin (BSA, Sigma-Aldrich) as a negative control. Serum samples were diluted between 1/200 and 1/800 in PBS (with 0.05 % Tween-20 + 5 % FCS) and used to probe duplicate wells. Wells were incubated with dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) europium-labeled anti-human IgG (Fcγ specific; Perkin Elmer, Waltham, MA, USA), and a Wallac Victor model 1420 Multilabel Counter was used to measure time-resolved fluorescence (Perkin Elmer). The BSA signal of each serum sample was subtracted from the signal for a given antigen. The B cell-activating factor (BAFF) ELISA was performed according to a previously described protocol [20 (link)].