Characterization of ERK5 Signaling Pathway

Cell culture reagents were purchased from Invitrogen and Sigma. Compounds 25 and 26 (ERK5-IN-1) were originally provided by Professor Nathanael Gray and Dr. Jinhua Wang, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA then latterly compound 26 was purchased from Selleckchem. AX15836 and JQ1 are from Tocris. BIX02189 is from Selleckchem. Anti-HA antibody for immunoblots was provided by the Babraham Institute Monoclonal Facility^{40 (link),41 (link)} and anti-HA (12CA5) antibody for immuno-fluorescence^{68 (link)} was from Roche/Sigma. Phospho-ERK5 TEY (3371) antibody was from Cell Signaling Technology^{40 (link),41 (link)}. Phospho-T733 and phospho-S754 ERK5 antibodies were a kind gift from Dr. Atanasio Pandiella, University of Salamanca, Spain^{16 (link)}. Anti-Flag antibody (M2) is from Sigma^{69 (link)}. Anti-MEK5 antibody (AB3184) is from EMD Millipore^{22 (link)}, anti-lamin A/C (636)^{70 (link)}, ERK5 (C-7)^{26 (link)} and myc (9E10)^{71 (link)} antibodies are from Santa Cruz and anti-MEK1/2^{72 (link)}, phospho-T359 RSK^{73 (link)} and total RSK^{74 (link)} from Cell Signaling Technology. Secondary antibodies were from BioRad^{40 (link),41 (link)}.

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Lochhead P.A., Tucker J.A., Tatum N.J., Wang J., Oxley D., Kidger A.M., Johnson V.P., Cassidy M.A., Gray N.S., Noble M.E, & Cook S.J. (2020). Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors. Nature Communications, 11, 1383.

Publication 2020

Cell culture reagents AX15836 BIX02189 Anti-Flag antibody (M2) anti-lamin A/C (636) myc (9E10)

Anti antibody Antibodies Antibody Bix02189 Cell culture Compound 26 Erk5 Immunoblots Lamin a c Mek1

Corresponding Organization : Babraham Institute

Other organizations : University of York, Newcastle University, Dana-Farber Cancer Institute

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Variable analysis

independent variables

Compounds 25 and 26 (ERK5-IN-1)

dependent variables

Phosphorylation of ERK5 at the TEY motif
Phosphorylation of ERK5 at T733 and S754
Expression of MEK5, ERK5, RSK, and other relevant proteins

control variables

Cell culture reagents
Antibodies (anti-HA, phospho-ERK5 TEY, phospho-T733 and phospho-S754 ERK5, anti-Flag, anti-MEK5, anti-lamin A/C, anti-ERK5, anti-myc, anti-MEK1/2, phospho-T359 RSK, total RSK)
Secondary antibodies

positive controls

Not explicitly specified

negative controls

Not explicitly specified

Annotations

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