Western Blotting Analysis of Signaling Pathways

Western blotting was performed as previously described39 (link). The conditions and duration of treatments of the cells before protein isolation are described in the text and the figure legends. Primary antibodies included p-ERK Thr204/205, ERK, p-MEK Ser217/221, MEK, anti-HA (clone C29F4) and GAPDH (all from Cell Signaling Technology, Danvers, MA), BRAF (H-145) and CRAF (E-10) (both from Santa Cruz biotechnology, Santa Cruz, CA), anti-MYC and anti-actin (Sigma Aldrich, St. Louis, MO). Secondary antibodies were HRP conjugates (Santa Cruz biotechnology, Santa Cruz, CA). Densitometry of bands was performed by using ImageJ program.

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Atefi M., Titz B., Tsoi J., Avramis E., Le A., Ng C., Lomova A., Lassen A., Friedman M., Chmielowski B., Ribas A, & Graeber T.G. (2016). CRAF R391W is a melanoma driver oncogene. Scientific Reports, 6, 27454.

Publication 2016

p-ERK Thr204/205 p-MEK Ser217/221 anti-HA (clone C29F4) GAPDH anti-MYC anti-actin HRP conjugates

Actin Antibodies Braf Cells Clone Craf Densitometry Gapdh Isolation Protein

Corresponding Organization :

Other organizations : University of California, Los Angeles, New York University, Institute for Molecular Medicine

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Variable analysis

independent variables

Treatments of the cells before protein isolation

dependent variables

Protein expression levels of p-ERK Thr204/205, ERK, p-MEK Ser217/221, MEK, BRAF, CRAF, MYC, actin, and GAPDH

control variables

Experimental conditions and procedures as previously described in reference 39 (link)

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

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