K ras f234 sc 30

Antibodies were obtained from the following sources: SOS1 (clone 25/SOS1), p120RasGAP (clone 13/RAS-GAP), MEK1 (no. 610121), MEK2 (no. 610235) were from BD Transduction Laboratories; K-Ras F234 (sc-30), N-Ras F155 (sc-31), pan-Ras C-4 (sc-166691), p-ERK1/2(Y204) (sc-101761), and Neurofibromin (sc-67) from Santa Cruz Biotechnology (Heidelberg, Germany); Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) (no. 2325), p44/42 MAPK (ERK1/2) (no. 4695), Akt (no. 9272), p-Akt(S473) (no. 4060), EGFR (no. 4267), p-EGFR (Y1068) (no. 2236), Phospho-p90RSK (Ser380) (no. 9341), RSK1/RSK2/RSK3 (32D7) (no. 9355), p-GSK-3β (Ser9) (D85E12) (no. 5558); GSK-3β (27C10) (no. 9315), Phospho-S6 Ribosomal Protein (Ser235/236) (no. 2211), S6 Ribosomal Protein (5G10) (no .2217), p70 S6 Kinase (49D7) (no. 2708) were from Cell Signaling Technology (Danvers, USA). Anti-DAB2IP (ab87811) was from Abcam (Cambridge, UK). Y13-259 rat monoclonal anti-Ras IP-antibody was purified from hybridoma supernatant (A.T.C.C., Manassas, U.S.A.).

Lysates were prepared from pellets using RIPA buffer and sonication, following standard protocols. Western blots were run on precast Any KD gels (Bio-Rad Labororatories Co., Ltd., Hercules, CA, USA) in accordance with standard protocols and transferred using the Bio-Rad Turbo blotter high molecular weight program. K-Ras (F234, sc-30, 1:500), N-Ras (F155, sc-31, 1:1000), Cyclin A (H-432, sc-751, 1:1000), B (H-433, sc-752, 1:1000), and E (C-19, sc198, 1:1000) were all purchased from Santa Cruz Biotechnology (Heidelberg, Germany) and Cyclin D (ab137875, 1:1000) was purchased from AbCam (Cambridge, UK). RNA-induced silencing complex (RISC) free control was from GE healthcare/Dharmacon Lafayette, CO, USA (D-001600-01-05). The membranes were blocked in 5% milk Phosphate Buffered Saline with 0.1% Tween (PBST 0.1%) for three hours before being incubated with primary antibody in 5% milk, PBST 0.1% at 4 °C overnight. Six ten-minute washes in PBST 0.1% were followed by incubation of the membrane with an HRP conjugated secondary anti-mouse antibody (sc-2357). Following incubation with a secondary antibody, six ten-minute washes with PBST 0.1% were carried out, and the membrane was visualised using chemo-luminescence and developed (Western lightning plus ECL Life Technologies, Paisley #31985-062).