Western Blot Analysis of Signaling Proteins

Western blots were performed as previously described, with antibodies for Ref-1 (mouse, 13B8E5C2, Novus Biologicals, Centennial, CO, USA), HIF1-α (rabbit, GTX127309, GeneTex, Irvine, CA, USA), p-STAT3 (Y705, rabbit, D3A7), STAT3 (mouse, 124H6), Cleaved PARP (poly-ADP ribose polymerase, rabbit, D64E10), total PARP (rabbit, #9542) (Cell Signaling, Danvers, MA, USA), Vinculin (mouse, V284, Sigma, St. Louis, MO, USA) and Actin (mouse, ACTN05(C4), NeoMarkers) at 1:1000 dilution.^{13 (link)–15 (link),28 (link)} For the HIF westerns, a urea lysis buffer (Tris, pH 6.8, 20 mM; NaH₂PO₄, 100 mM; urea, 6 M) was used for protein extraction.

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Gampala S., Shah F., Zhang C., Rhodes S.D., Babb O., Grimard M., Wireman R.S., Rad E., Calver B., Bai R.Y., Staedtke V., Hulsey E.L., Saadatzadeh M.R., Pollok K.E., Tong Y., Smith A.E., Clapp D.W., Tee A.R., Kelley M.R, & Fishel M.L. (2021). Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours. British Journal of Cancer, 124(9), 1566-1580.

Publication 2021

p-STAT3 Cleaved PARP total PARP Vinculin

Actin Antibodies Biologicals Buffer Dilution Hif1 α Mouse Novus Poly adp ribose polymerase Protein Rabbit Ref 1 Stat3 Tris Urea Vinculin Western blots

Corresponding Organization :

Other organizations : Indiana University – Purdue University Indianapolis, Cardiff University, Johns Hopkins University, Johns Hopkins Medicine

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Variable analysis

independent variables

Antibodies used for Western blot analysis, including Ref-1, HIF1-α, p-STAT3, STAT3, Cleaved PARP, total PARP, Vinculin, and Actin

dependent variables

Protein expression levels of Ref-1, HIF1-α, p-STAT3, STAT3, Cleaved PARP, total PARP, Vinculin, and Actin

control variables

Protein extraction method (urea lysis buffer for HIF westerns)
Antibody dilution (1:1000)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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