Protein expression analysis by Western blot

Western blot and immunohistochemical analyses were performed as described previously.20 (link),49 (link) Primary antibodies against PBF (custom20 (link),49 (link)), HA (MMS-101P; BioLegend, San Diego, CA, USA), hPTTG (700791; Invitrogen, Paisley, UK), phospho-p44/42 MAPK (Erk1/2) (9101), p44/42 MAPK (Erk1/2) (4695), Myc-Tag (2276; Cell Signaling Technology, Danvars, MA, USA), p53 (sc-126; Santa Cruz Biotechnology, Dallas, TX, USA), cyclin D1 (ab16663), cyclin A2 (ab181591; Abcam, Cambridge, UK), Chk1 (GTX50463), Brca1 (GTX50557; GeneTex, Irvine, CA, USA), phospho-CHK1(Ser345) (PA5-34625; Thermofisher Scientific, Waltham, MA, USA) and β-actin (A5441; Sigma-Aldrich, Dorset, UK) were used. Densitometry was performed on blots scanned into Photoshop (Adobe Systems) and analysed using ImageJ as described.27 (link)

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Read M.L., Fong J.C., Modasia B., Fletcher A., Imruetaicharoenchoke W., Thompson R., Nieto H., Reynolds J.J., Bacon A., Mallick U., Hackshaw A., Watkinson J.C., Boelaert K., Turnell A.S., Smith V.E, & McCabe C.J. (2017). Elevated PTTG and PBF predicts poor patient outcome and modulates DNA damage response genes in thyroid cancer. Oncogene, 36(37), 5296-5308.

Publication 2017

MMS-101P hPTTG p44/42 MAPK Myc-Tag (2276; p53 (sc-126; ab16663 ab181591 phospho-CHK1(Ser345) β-actin

Actin Antibodies Brca1 Cyclin a2 Cyclin d1 Densitometry Erk1 Hpttg Western blot

Corresponding Organization :

Other organizations : University of Birmingham, Freeman Hospital, University College London, Cancer Research UK, University Hospitals Birmingham NHS Foundation Trust

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Variable analysis

independent variables

Primary antibodies against PBF (custom, references 20 and 49)
Primary antibodies against HA (MMS-101P; BioLegend, San Diego, CA, USA)
Primary antibodies against hPTTG (700791; Invitrogen, Paisley, UK)
Primary antibodies against phospho-p44/42 MAPK (Erk1/2) (9101)
Primary antibodies against p44/42 MAPK (Erk1/2) (4695)
Primary antibodies against Myc-Tag (2276; Cell Signaling Technology, Danvars, MA, USA)
Primary antibodies against p53 (sc-126; Santa Cruz Biotechnology, Dallas, TX, USA)
Primary antibodies against cyclin D1 (ab16663)
Primary antibodies against cyclin A2 (ab181591; Abcam, Cambridge, UK)
Primary antibodies against Chk1 (GTX50463)
Primary antibodies against Brca1 (GTX50557; GeneTex, Irvine, CA, USA)
Primary antibodies against phospho-CHK1(Ser345) (PA5-34625; Thermofisher Scientific, Waltham, MA, USA)
Primary antibodies against β-actin (A5441; Sigma-Aldrich, Dorset, UK)

dependent variables

Western blot analysis
Immunohistochemical analysis

control variables

Densitometry was performed on blots scanned into Photoshop (Adobe Systems) and analysed using ImageJ as described (reference 27)

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