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2 protocols using ab50247

1

Analyzing Autoantibody Reactivity in Rheumatoid Arthritis

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Samples were dotted onto NCMs at 10 ng/dot and blocked in 5% goat serum (Sigma-Aldrich) in TBS. Blots were incubated in either samples (RA SF or PB serum, NL PB serum, 1:10,000), normal human IgG control (R&D Systems, #1001A, 1 μg/mL), or control antibodies, including monoclonal mouse anti-ID1 (Abcam, #ab66495, clone 2456C1a, 1 μg/mL) and polyclonal rabbit anti-PAD4 (Abcam, #ab50247, 1 μg/mL), then probed with peroxidase AffiniPure goat anti-human IgG (Jackson ImmunoResearch, #109035003, 1:5,000). To verify specificity for citID1 reactivity of RA specimens, additional control blots were included on the same NCMs using various proteins, including noncitID1, BSA, citBSA, ENA-78, citENA-78 and rhPAD4. Densitometry analysis was performed using ImageJ.
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2

Protein Interactions and Modifications Assessment

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PAD4 (ab128086 and ab50247, Abcam), HA (MMS-101R, Covance), Flag (F3165, Sigma), AMC antibody kit (17-347, Millipore), E2F-1 (KH95 and C20, Santa Cruz Biotechnology), BRD4 (ab128874, Abcam), β-actin (A2228, Sigma), pRB (IF8, Santa Cruz Biotechnology), CD11B (ab75476, Abcam), mouse and rabbit secondary antibodies (GE Healthcare), and HA or Flag antibody-coupled agarose beads (Sigma) were used.
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