Src kinase

24 h after plating, 50% confluent cells were treated. The drug concentrations applied are listed in Table 3 (single treatment) respectively Table 4 (combination treatment). After 6 h, 24 h or 72 h for single or combination treatment, respectively at day 8 of the multimodal RIST and TIST treatment, the cells were harvested. The whole cell lysates were separated by gel electrophoresis and the proteins were transferred to nitrocellulose membranes (Amersham^TM Protran^TM 0,45 µm, GE Healthcare Life Science, Cat. No. 10600002). After the transfer, the membranes were incubated with primary antibodies (1:1000, respectively 1:2000 for LC3B and GAPDH antibody). The primary antibodies AKT (pan, #4691), phospho-AKT (Ser473, #4060), phospho-AKT (Thr308, #13038), 4E-BP1 (#9644), phospho-4E-BP1 (Ser65, #9456), MycN (#9405), p70S6 kinase (#9202), phospho-p70S6 kinase (Thr389, #9234), Src kinase (#2108), phospho-Src family (Tyr416, #2101), caspase-3 (#9665) and p21 Waf1/Cip (DCS60, #2946) were purchased from Cell Signaling. The LC3B antibody (#NB100-2220SS) was purchased from NovusBio, the PARP-1 antibody (#1072-1) was purchased from Epitomics and the GAPDH antibody (#Sc-47724) was purchased from Santa Cruz. ImageJ was used for quantification of the protein expression.

Fetal bovine serum, penicillin/streptomycin, and DMEM were purchased from Invitrogen. PAO, rapamycin, and SP600125 were obtained from Calbiochem, and [3H]-palmitic acid was from Perkin Elmer Life Sciences. 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphate (PA) dissolved in chloroform was purchased from Avanti Polar Lipids (Alabaster, AL, USA). The following polyclonal antibodies from Cell Signaling (Beverly, MA, USA) were used: PLD1, PLCγ, p-PLCγ, Src kinase, p-Src kinase, mTOR, p-mTOR, p-p70S6K antibody, and p70S6K. GAPDH and PLCγ were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell lysates and electrophoresis was performed as previously described [7 (link)]. Immunoblotting of Src (Y⁴¹⁹; Millipore, USA) [14 (link)], Src kinase (Cell Signaling Technology, USA) [15 ], FAK (Y⁸⁶¹; Invitrogen, UK) [16 (link)] and FAK (Y³⁹⁷; Abcam, UK) [17 (link)] were performed at 1:1000 dilutions with rabbit anti-human antibodies. Proteins were visualized by addition of a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit IgG. HRP-conjugated antibodies raised against beta tubulin were used as loading controls. HRP-conjugated protein was visualized using an enzyme-linked chemiluminescence reaction (Thermo Fisher Scientific, UK) and quantified using a LAS3000 image analyser (Fujifilm, Tokyo) and analysed with Image J software (NIH, US).