Western Blot Analysis of Iron Homeostasis

Cells were lysed with NP-40 lysis buffer (25 mM tris (pH 7.4), 1% Triton X-100, 1% SDS, 1% sodium deoxy-cholate, 150 mM NaCl, aprotinin (2 μg/ml), 1 mM phenylmethylsulfonyl fluoride) containing complete protease inhibitor cocktail (Roche Diagnostics). Samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes before being incubated with primary and secondary antibodies. Bands were detected using chemiluminescence (Thermo Scientific). Membranes were probed with antibodies to β-actin (Sigma, Cat# A3854 ), ferroportin (Novus Biologicals, cat# NBP121502 ), transferrin receptor1 (Invitrogen, cat# 13-6890), IRP2 (Santa Cruz, cat# sc-33682), ferritin H (17 (link)), p-STAT3 (cell Signaling, cat# 9131S) and STAT3 (cell Signaling, cat#4904S ). Western blots were quantified using ImageJ software.

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Basuli D., Tesfay L., Deng Z., Paul B., Yamamoto Y., Ning G., Xian W., McKeon F., Lynch M., Crum C.P., Hegde P., Brewer M., Wang X., Miller L.D., Dyment N., Torti F.M, & Torti S.V. (2017). Iron addiction: A novel therapeutic target in ovarian cancer. Oncogene, 36(29), 4089-4099.

Publication 2017

chemiluminescence β-actin ferroportin transferrin receptor1 p-STAT3 STAT3

Actin Antibodies Aprotinin Biologicals Buffer Cells Chemiluminescence Deoxy Diagnostics Ferritin Ferroportin Membranes Nacl Nitrocellulose Novus Np 40 Phenylmethylsulfonyl fluoride Protease inhibitor Sds polyacrylamide gel electrophoresis Sodium cholate Stat3 Transferrin Tris Triton x 100 Western blots

Corresponding Organization :

Other organizations : UConn Health, Jackson Laboratory, The University of Texas Health Science Center at Houston, University of Houston, Brigham and Women's Hospital, Wake Forest University

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Variable analysis

independent variables

Lysis buffer composition (25 mM tris (pH 7.4), 1% Triton X-100, 1% SDS, 1% sodium deoxy-cholate, 150 mM NaCl, aprotinin (2 μg/ml), 1 mM phenylmethylsulfonyl fluoride)

dependent variables

Protein expression levels of β-actin, ferroportin, transferrin receptor1, IRP2, ferritin H, p-STAT3, and STAT3

control variables

Complete protease inhibitor cocktail (Roche Diagnostics)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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