Go6976

GO6976 (GO), LY333531 (LY), Rottlerin (Rott), rosmarinic acid, and apigenin were purchased from Sigma Chemical Co. (Yongin, Korea). Salvianolic acid B was purchased from Santa Cruz (Santa Cruz, CA, USA). Stock solutions of GO, LY, Rott, and apigenin were dissolved in dimethyl sulfoxide (DMSO). Salvianolic acid B and rosmarinic acid were dissolved in distilled water. The final concentration of DMSO was less than 0.1%.

Swiss3T3 and LA4 cells were obtained from ATCC (Manassas, VA, USA). LKR murine lung cells are generous gift from Dr. Jacks (MIT, Cambridge) and have been used in various studies [49 (link)–51 (link)]. The cells were cultured in DMEM medium containing 10% fetal calf serum. SK1 cells are Swiss3T3 cells stably transfected with v-K-ras and maintained in the growth medium containing G418 (200 μg/ml). GO6976 was purchased from Sigma. Bcl-2 mutants were inserted in the pEX-GST expression vector and obtained from Dr. Luo (Boston University).

MCF10A and MCF10A Rictor^ZFN were purchased from Sigma-Aldrich and cultured in Growth Medium [DMEM:F12 supplemented with 5% Horse Serum (Life Technologies), 10 μg/ml porcine insulin (Sigma-Aldrich), 20 nanograms/ml human epidermal growth factor (R&D Systems), 10 nanograms/ml cholera toxin (Sigma-Aldrich), 100 micrograms/ml hydrocortisone (Sigma-Aldrich), 100 I.U./ml penicillin-streptomycin (Life Technologies)]. For some experiments, cells were maintained for 24 hour in Starvation Media [Growth Media without serum or EGF] prior to stimulation and/or analysis. PKC-alpha inhibitor GO6976 (Sigma-Aldrich, 2 nm) and adenoviral particles (Ad.RFP and Ad.PKC-alpha, Vector Biolabs) were purchased. Cells were incubated with adenoviral particles (5 X 10⁸ particle forming units/ml) for 3–5 hours at 37°C and cells were allowed to recover for 48 hours prior to experimental analysis.

Quiescent cells (2 × 10⁵) were stimulated for 24 to 48 h in a U-bottom 96-well plate in the presence of either 1 TCR Dynabead per cell (Life Technologies), 500 nM SAHA (Caymen Chemical), 10 ng/ml TNF-α, 10 ng/ml IL-15, or 5 ng/ml IL-7 (all from Peprotech). For the transcription inhibitor panel, 2 × 10⁵ cells were pretreated for 2 h prior to stimulation with one of the following: 1 µM cyclosporine (Sigma-Aldrich), 1 µM Go6976 (Sigma-Aldrich), or 1 µM IKK inhibitor IV (Santa Cruz Biotechnologies).

PcDNA SH-SY5Y, TrkA SH-SY5Y, and TrkAIII SH-SY5Y cell lines were grown in recommended medium (RPMI or Dulbecco's modified Eagle's medium) supplemented with 10% foetal calf serum and appropriate antibiotics (phleomycin [Zeocin], penicillin, and streptomycin), as described previously [1 (link)-4 ]. Cell culture reagents, MG-132, geldanamycin, cytochalasin D, nocodozol, proteinase inhibitor Cocktail, Bis-benzimide tri-hydrochloride, propidium iodide, Histodenz^TM, NGF, K252a, Go6976 and GW441756 were purchased from Sigma-Aldrich (St. Louis MO). Endoglycosidase H was from Roche Italia (Milan, It). The pan Trk inhibitor CEP-701 was from Cephalon Inc. (West Chester, PA). Polyclonal anti-TrkA (C14) and monoclonal anti-Phospho-tyrosine (pY99) antibodies were from SantaCruz (Santa Cruz, Ca). The polyclonal anti-α-tubulin antibody was from Sigma (St. Louis, MO). Polyclonal anti-phospho-Y674/675 TrkA, anti-COPI, COPII and mouse monoclonal anti-Calnexin and anti-TGN46 antibodies were from Abcam (Cambridge, UK). The polyclonal anti-phospho-Y490 TrkA antibody was from Cell signalling (Danvers, MA). Mouse monoclonal anti-GM130 antibody was from BD Bioscience (San Jose, Ca). FITC and TRIC-conjugated secondary anti mouse and anti-rabbit IgG antibodies were from Jackson Immune Research (Bar Harbor, Maine). VectorMount^TM medium was from Vector Laboratories (Berlingame, Ca).