Phospho mapk erk1 2

Commercial antibodies included: TrkB (#610101, mouse, BD Biosciences, San Jose, CA); phospho-TrkB Tyr515 (#9141, rabbit, Cell Signaling, Danvers, MA) and Tyr705/706 (#4621, rabbit, Cell Signaling), phospho-Akt (#4051, mouse, Cell Signaling), Akt (#4691, rabbit, Cell Signaling), phospho-CREB (#9198, rabbit, Cell Signaling), CREB (#9104, mouse, Cell Signaling), phospho-MAPK (Erk1/2) (#9106, mouse, Cell Signaling), ERK (#sc-154, rabbit, Santa Cruz, Santa Cruz, CA), FLAG (#F7425, rabbit, Sigma), GAPDH (MAB374, mouse, EMD Millipore) and MAP2 (AB15452, chicken, EMD Millipore). The Myc monoclonal 9E10 was from spent hybridoma medium (Borjigin and Nathans, 1994 (link)). Rabbit antibody to the Kalirin Sec14 domain has been described and validated (CT302) (Yan et al., 2015 (link)).

MDA-MB-231 breast cancer cells were serum starved for 6 hours and stimulated with 1 nM human β-endorphin (Fisher Scientific, Waltham, MA) or sterile PBS. Cell lysates were prepared as previously described using a cocktail of protease inhibitors. [23 (link)] Protein lysates containing 30 μg of protein were separated on a 3–15% gradient SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane (Immobilon; Millipore, Bedford, MA). Protein bands were detected using 1:500 phospho-Akt, 1:500 Akt, 1:1000 phospho-STAT3, 1:1000 STAT3, 1:1000 phospho-MAPK/ERK1/2, and 1:1000 MAPK/ERK1/2; all from Cell Signaling Technology, Danvers, MA). Alkaline phosphatase-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch, West Grove, PA) and ECF system (Amersham Bioscience, Buckinghamshire, UK) were used to detect chemiluminescent signals on a Storm 860 Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Protein bands were quantitated by densitometric analysis using ImageJ Software (National Institutes of Health, Bethesda, MD).