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3 protocols using cxcl13

1

Signaling Pathway Inhibitor Protocol

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We purchased p-PLCβ, PLCβ, p-PKCα, PKCα, p-c-Src, c-Src, p-p65, p65, CXCL13, VCAM-1, p-IKK, IKK, p-IκBα, IκBα, and actin from GeneTex International Corporation (Hsinchu City, Taiwan). All ON-TARGETplus siRNAs were purchased from Dharmacon (Lafayette, CO, USA). CXCR5 mAb was purchased from R&D Systems (Minneapolis, MN, USA). Cell culture supplements were purchased from Invitrogen (Carlsbad, CA, USA). A Dual-Luciferase® Reporter Assay System was bought from Promega (Madison, WI, USA). qPCR primers and probes, as well as the Taqman® One-Step PCR Master Mix, were supplied by Applied Biosystems (Foster City, CA, USA). The PLCβ inhibitor (U73122), PKCα inhibitor (GF109203X), c-Src inhibitor (PP2), IκB inhibitor (TPCK), NF-κB inhibitor (PDTC), NF-κB inhibitor (BAY 11-7082), and other chemicals not already mentioned were supplied by Sigma-Aldrich (St. Louis, MO, USA).
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2

Molecular Profiling of Inflammatory Signaling

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Anti‐mouse and anti‐rabbit IgG‐conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for PLCβ3 (Santa Cruz, sc‐403), PKCα(Santa Cruz, sc‐208), c‐Src (cell signaling, #2109s), p65 (Santa Cruz, sc‐109), IκBα (Santa Cruz, sc‐1643), p‐PLCβ (cell signaling, #2481s), p‐PKCα(cell signaling, #9375), p‐c‐Src (cell signaling, #5473), p‐p65 (cell signaling, #3033), p‐IKKα/β (cell signaling, #2694), IKKα/β (Santa Cruz, sc‐7218), p‐IκBα (cell signaling, #9246), CXCL13 (GeneTex, GTX108471), CXCR5 (GeneTex, GTX100351), VCAM‐1 (GeneTex, GTX110684) and β‐Actin (GeneTex, GTX109639) were purchased from Cell Signaling Technology, Inc, Santa Cruz Biotechnology, Inc and GeneTex Inc. All other chemicals were obtained from Sigma‐Aldrich. The CXCL13 shRNA plasmid was purchase from RNAiCore (Clone ID: TRCN0000057983). All siRNAs were purchased from Sigma‐Aldrich (Mission® siRNA).
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3

Immunostaining of Lung Tissue Sections

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Frozen-embedded sections (lungs) from wildtype and Abcg1−/− mice were fixed in 4% PFA, blocked with 5% goat serum, and stained with either HRP-conjugated anti-mouse IgM, IgG or IgA and detected with ECL. A Vectastain ABC-Alkaline phosphatase kit (Vector Laboratories) was used to visualize the antibody staining. Where indicated, slides were counter-stained with Harris Hematoxylin (Fisher Scientific). Frozen tissue sections of lungs from wildtype and Abcg1−/− mice were also stained with antibodies that recognize CXCL13 (Genetex), oxPL (E06), B220 (B cell marker; BD Biosciences Clone RA3–6B2) and PCNA (proliferative marker; Genetex), followed by anti-mouse IgM AlexaFluor 488, anti-rat AlexaFluor 594 or anti-rabbit AlexaFluor 488 secondary antibodies (Molecular Probes, Life Sciences). Immunostaining of adjacent sections in the absence of primary antibody was used as a negative control.
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