Immunoblotting Assay for Cell Signaling

Cells lysates were harvested by 2% SDS lysis buffer, heated and sonicated. The protein concentrations of the cell lysates were estimated using a Pierce^® BCA Protein Assay kit. The detailed protocol used for immunoblotting can be found in our previous report [9 (link)]. The primary and secondary antibodies used in the current study were listed as follows: p53 (DO-7) (#M7001, Dako, Glostrup, Denmark), MDM2 (Ab-1) (#OP46, Merck Millipore, Watford, UK), p21^WAF1 (EA10) (#OP64, Calbiochem (Millipore, Watford, Herts, UK), p-ERK (E-4) (#sc-7383, Santa Cruz, Dallas, TX, USA), ERK (K-23) (#sc-94, Santa Cruz), GAPDH (14C10) (#2118, Cell Signaling Technology, Danvers, MA, USA), WIP1 (F-10) (#sc-376257, Santa Cruz Biotechnology), phospho-p53 (S15) (ab1431, Abcam, Cambridge, UK), phospho-ATM(S1981) (#AF1655, R&D systems, Abingdon, UK), actin (#A4700, Sigma-Aldrich), secondary goat anti-mouse/rabbit horseradish peroxidase (HRP)-conjugated antibodies (#P0447/P0448, Dako).

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Wu C.E., Koay T.S., Esfandiari A., Ho Y.H., Lovat P, & Lunec J. (2018). ATM Dependent DUSP6 Modulation of p53 Involved in Synergistic Targeting of MAPK and p53 Pathways with Trametinib and MDM2 Inhibitors in Cutaneous Melanoma. Cancers, 11(1), 3.

Publication 2018

p53 (DO-7) MDM2 (Ab-1) p-ERK (E-4) ERK (K-23) GAPDH (14C10)

Actin Anti antibodies Antibodies Assay Buffer Gapdh Goat Horseradish peroxidase Mdm2 Mouse Protein Rabbit

Corresponding Organization : Newcastle University

Other organizations : Linkou Chang Gung Memorial Hospital, Chang Gung University, University College London, Cancer Research UK

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of p53, MDM2, p21WAF1, p-ERK, ERK, GAPDH, WIP1, phospho-p53 (S15), phospho-ATM(S1981), actin

control variables

2% SDS lysis buffer used to harvest cell lysates
Pierce® BCA Protein Assay kit used to estimate protein concentrations
Detailed immunoblotting protocol from previous report [9]
Primary and secondary antibodies used for immunoblotting

controls

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

Annotations

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