Total akt

Samples were collected and prepared as described previously (Ko et al. 2001 (link), 2007 (link)). Briefly, cultured cells were washed in ice-cold phosphate-buffered saline and lysed in radio immunoprecipitation assay buffer. In some experiments, intact retinas were homogenized in radio immunoprecipitation assay buffer. The samples were separated on 10% sodium dodecyl sulfate—polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in the studies were anti-VGCCα1D (Alomone, Jerusalem, Israel), anti-phospho308 Akt (pAktThr308; Cell Signaling Technology, Danvers, MA, USA), anti-phospho473 Akt (pAktSer473; Cell Signaling Technology), a polyclonal antibody insensitive to the phosphorylation state of Akt (total Akt, used for loading control; Cell Signaling Technology), a monoclonal antibody specific for di-phospho-Erk (pErk; Sigma, St Louis, MO, USA), and a polyclonal antibody insensitive to the phosphorylation state of Erk (total Erk, used for internal control and loading control; Santa Cruz Biochemicals, Santa Cruz, CA, USA). Blots were visualized using appropriate secondary antibodies conjugated to horse radish peroxidase (Cell Signaling Technology) and an ECL detection system (Pierce, Rockford, IL, USA). For the cell-surface biotinylation assays, cultured cells were washed and exposed to sulfo-NHS-LC-biotin at 4°C for 30 min using a commercially available cell-surface biotinylation assay kit (Pierce). A portion of the whole-cell lysate was used to measure the VGCCα1D subunit and total Erk. The rest was used to determine plasma membrane-bound VGCCα1D subunits by incubating it with streptavidin-linked agarose beads. The beads were collected, and the amount of VGCCα1D subunit was determined by immunoblotting. The ratio of VGCCα1D subunit to total Erk, pAktThr308 to total Erk, and pAktSer473 to total Erk in each sample was determined by densitometry using Scion Image (NIH, Bethesda, MD, USA). Previously, we showed that there is a circadian regulation of pErk in chick retinae (Ko et al. 2001 (link), 2007 (link)). We used the ratio of pErk/total Erk as our internal control of circadian timing (data not shown). All measurements were repeated five to six times. PD 98059 [mitogen-activated kinase kinase (MEK) inhibitor] and manumycin A (small GTPase inhibitor) were from A. G. Scientific (San Diego, CA, USA); LY 294002 (PI3K inhibitor) and 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor; Akti) were obtained from Calbiochem/EMD (San Diego, CA, USA).

HCC78 was a kind gift from John D. Minna. Ba/F3 cells were a kind gift from Dan Theodorescu. 293T and NIH3T3 cells were purchased from ATCC (Manassas, VA). Crizotinib (PF-02341066) and PF-04217903 were obtained from Pfizer Inc. (New York, NY). NVP-TAE684 and gefitinib were purchased from Selleck Chemicals (Houston, TX). Antibodies used were as follows: ROS1 pY2274 (3078, Cell Signaling, Danvers, MA), total ROS1 (sc-6347, Santa Cruz Biotechnology, Santa Cruz, CA), SHP-2 pY542 (3751, Cell Signaling), total SHP-2 (610621, BD Biosciences), AKT pS437 (4058, Cell Signaling), total AKT (2920, Cell Signaling), STAT3 pY705 (9145, Cell Signaling), total STAT3 (9139, Cell Signaling), ERK pT202/Y204 (9101, Cell Signaling), total ERK (9107, Cell Signaling), α-tubulin (sc-8035, Santa Cruz Biotechnology), and GAPDH (MAB274, Millipore).