Quantifying Phosphorylated MEK and ERK

CD138⁺ cells were lysed with M-Per buffer (Thermo Fisher Scientific, Rockford, IL) containing phosphatase and protease inhibitors. Approximately 50–72 ng of protein was used per sample, and experiments were performed using a Nanopro1000 instrument (ProteinSimple, Santa Clara, CA), as previously described (29 (link), 30 (link)). Various phosphorylated isoforms of MEK and ERK were detected using anti-phospho-ERK (Cell Signaling Technology, Danvers, MA), anti-MEKpS218/222 (Epitomics, Burlingame, CA), anti-MEKpT292 (Millipore), anti-MEKpT386 (Novus Biologicals, Littleton, CO), anti-MEKpT394 (Abcam, Cambridge, MA), anti-MEKpS298 (Cell Signaling Technology), and anti-Beta2-microglobulin (Abcam) primary antibodies with HRP-conjugated goat anti-rabbit or goat anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies. Luminol and peroxide (ProteinSimple) were added to generate chemiluminescent light. The digital images were analyzed and quantified with Compass software (ProteinSimple). ERK and MEK were normalized to Beta2-microglobulin loading controls.

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Holkova B., Zingone A., Kmieciak M., Bose P., Badros A.Z., Voorhees P.M., Baz R., Korde N., Lin H.Y., Chen J.Q., Herrmann M., Xi L., Raffeld M., Zhao X., Wan W., Tombes M.B., Shrader E., Weir-Wiggins C., Sankala H., Hogan K.T., Doyle A., Annunziata C.M., Wellons M., Roberts J.D., Sullivan D., Landgren O, & Grant S. (2015). A Phase 2 Trial of AZD6244 (Selumetinib, ARRY-142886), an Oral MEK1/2 Inhibitor, in Relapsed/Refractory Multiple Myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(5), 1067-1075.

Publication 2015

M-Per buffer anti-phospho-ERK anti-Beta2-microglobulin HRP-conjugated goat anti-rabbit or goat anti-mouse

Anti antibodies Antibodies Biologicals Buffer Cd138 Cells Goat Isoforms Light Luminol Mouse Novus Peroxide Phosphatase Protease inhibitors Protein Rabbit

Corresponding Organization : Institute for Molecular Medicine

Other organizations : National Cancer Institute, National Institutes of Health, Center for Cancer Research, University of Maryland, Baltimore, University of North Carolina at Chapel Hill, Moffitt Cancer Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Phosphorylated isoforms of MEK and ERK

control variables

Protein loading controlled using Beta2-microglobulin

controls

Positive control: None mentioned
Negative control: None mentioned

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