Anti torc1 4e7 c1 f9 e6

Following stimulation, cells were washed with cold phosphate-buffered saline (PBS) and collected via scraping. Nuclear extracts were prepared using a NE-PER kit from Pierce (Rockford, IL). Protein concentration in the extracts was determined using the BCA protein assay kit (Pierce, Rockford, IL). The extracts were stored at −80 °C prior to use. Proteins (10 µg per lane) were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Western blotting was carried out using following primary antibodies according to manufacturer protocol: anti-CREB (06–863, Millipore, Billerica, MA), anti-pCREB (06–519, Millipore, Billerica, MA), anti-TORC1 (4E7-C1-F9-E6, GeneTex, Irvine, CA), anti-TORC2 (ab167129, Abcam, Cambridge, MA), anti-TORC3 (EPR3440, GeneTex, Irvine, CA), and anti-β-actin (#4967, Cell Signaling, Danvers, MA). Appropriate secondary antibodies conjugated with horseradish peroxidase were obtained from Jackson ImmunoResearch (West Grove, PA). Signals were visualized by chemiluminescence using ECL detection reagents (GE Healthcare, Piscataway, NJ), captured by ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA), quantified using ImageJ software [34] (link), and normalized to β-actin for comparison.