Western Blot Analysis of Cell Signaling

Cells lysates (20–40µg protein) were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad Laboratories, Hercules, CA,) and transferred onto a Polyvinylidene fluoride (PVDF) membranes (PerkinElmer Life Science, Waltham, MA) by semi-dry electroblotting. Membranes were probed with rabbit anti-human A20/TNFAIP3 (Abcam), Ser-536 Phospho-P65 (Cell Signaling, Danvers, MA), Ser-32 phospho-IκBα, (Cell Signaling) and, GAPDH (Calbiochem/ EMD Biosciences, Gibbstown, NJ) followed by appropriate secondary HRP-conjugated antibodies (Thermo Scientific, Rockford, IL). Protein bands were detected with an enhanced chemiluminescence kit (PerkinElmer Life Science) followed by exposure to an autoradiography film(5 (link), 9 (link)).

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Moll H.P., Lee A., Peterson C.R., Cervantes J.R., Wojcik B.M., Parulkhar A., Mele A., LoGerfo P.J., Siracuse J.J., Csizmadia E., da Silva C.G, & Ferran C. (2016). A20 Haploinsufficiency Aggravates Transplant Arteriosclerosis in Mouse Vascular Allografts: Implications for Clinical Transplantation. Transplantation, 100(11), e106-e116.

Publication 2016

SDS-PAGE PVDF) membranes HRP-conjugated antibodies enhanced chemiluminescence kit

Antibodies Autoradiography Cells Chemiluminescence Gapdh Human Membranes Phospho Polyvinylidene fluoride Protein Rabbit Sds page Tnfaip3

Corresponding Organization : Beth Israel Deaconess Medical Center

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Variable analysis

independent variables

Cell lysates (20–40µg protein)

dependent variables

Protein bands (detected with enhanced chemiluminescence kit)

control variables

Reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Transfer onto Polyvinylidene fluoride (PVDF) membranes by semi-dry electroblotting
Probing with rabbit anti-human A20/TNFAIP3, Ser-536 Phospho-P65, Ser-32 phospho-IκBα, and GAPDH antibodies
Appropriate secondary HRP-conjugated antibodies

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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