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2 protocols using ifit3

1

Immunohistochemical Staining of IFIT3 and GBP2

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LSG paraffin sections from SS patients were processed for immunohistochemistry as described (12 (link)). Briefly, after rehydration, antigen retrieval and blocking, sections were incubated overnight at 4°C with antibodies against IFIT3 (12.5 µg/ml, Novus Biologicals) or GBP2 (30 µg/ml; Novus Biologicals). HRP-conjugated secondary antibody incubations were performed for 1 hour at RT, and staining was visualized with diaminobenzidine (Dako) per the manufacturer’s directions. Nuclei were counterstained with Mayer’s hematoxylin. All images were captured using a Zeiss Axioskop 50 with a Zeiss AxioCam HRc camera and AxioVision 4 software.
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2

Immunoblot Analysis of Cellular Signaling

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Cells were lysed in NT2 buffer containing 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl2, 0.05% Nonidet P-40, 1 mmol/L sodium orthovanadate, 0.5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 2 μg/mL leupeptin, and 2 μg/mL pepstatin. Proteins from cell lysates were separated by 10% SDS-PAGE gel electrophoresis (Bio-Rad), and transferred to nitrocellulose membranes (Invitrogen). Immunoblots were probed with antibodies recognizing: Aiolos (Cell Signaling Technology), Ikaros (Millipore), IRF7 (Cell Signaling Technology), DDX58 (Thermo Scientific), IFIT3 (Novus Biologicals), c-myc (Abcam), IRF4 (Santa Cruz), p65 (Cell Signaling Technology), p-p65 Ser236 (Cell Signaling Technology), p105/p50 (Cell Signaling Technology), p100/p52 (Cell Signaling Technology), Tubulin (Cell Signaling Technology), TBP (Cell Signaling Technology), and β-actin (Li-Cor). Signals were detected with a Li-Cor Odyssey imager or WES (Protein Simple).
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