Pakt s473

Western blotting was performed using standard methods. After treatment with indicated drugs, cells were washed with cold PBS and lysed in the following lysis buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 50 mM NaF, 10 nM β-glycerophosphate, 1 mM sodium vanadate, 0.5 mM DTT, 4 μg/mL leupeptin, 4 μg/mL pepstatin, 4 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 16,000 × g for 5 min at 4°C. Protein concentrations were determined by BCA assay (Thermo Scientific). Proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham). Immunoblotting was performed per antibody manufacturer's specifications. Antibodies for MET, P-MET (Y1234/1235) HER2, P-HER2 (Y1221/1222), P-AKT (S473), and P-S6 (S240/244), (Cell Signaling) were used at 1:1000 dilution; P-ERK (Cell Signaling) was used at 1:2000 dilution. GAPDH (Millipore) was used at 1:1000 dilution. Protein detection on Western blots was performed using SuperSignal chemiluminescence (Thermo Scientific).

Anti-FLAG M2 (#F1804) and Anti-FLAG M2-HRP (#A8592) were purchased from Sigma-Aldrich and anti-Src antibody (#MA5-15120) was from Thermo Scientific. p-Src (Y416) (#2113), p-Abl (Y412) (#2865), p-Tyr-100 (#9411), p-Tyr-1000 (#8954), anti-GAPDH (#14C10), Myc Tag (mouse #2276 and rabbit #71D10), p-AKT (T308) (#13038), p-AKT (S473) (#4060), p70 S6 kinase (#9202), and phospho-p70 S6K (T389) (#9234) primary antibodies were purchased from Cell Signaling Technologies (CST). Secondary antibodies Goat anti-Mouse-HRP (#31430) and Goat anti-Rabbit-HRP (#31460) were from Thermo Scientific. Alexa Fluor 488 anti-mouse (#A11029) and Alexa Fluor 647 anti-rabbit (#A21245) were from Life Technologies. All primary antibodies were used at a 1:1000 dilution and secondaries at 1:5000. Protease inhibitor cocktail (#P8340), Phosphatase inhibitor cocktail 2 (#P0044) and 3 (#P5726) were purchased from Sigma-Aldrich. NeuCode labeling reagents L-Lysine:2HCl (3,3,4,4,5,5,6,6-D8, 98%) (#DLM-2641-0) and L-Lysine:2HCl (13C6, 99%, 15N2, 99%) (#CNLM-291-H-0.25) were from Cambridge Isotopes. Rapamycin was from Cayman Chemical and M-PER extraction reagent (#78501) was from Thermo Scientific. All restriction enzymes and DNA polymerases were purchased from NEB (Ipswich, MA). Oligonucleotides and gBlocks Gene Fragments were purchased from IDT and all constructs were verified by DNA sequencing (Quintara Biosciences).

Approximately, 30 to 80 mg of nuclear or cytoplasmic protein that was isolated from SW480 colorectal cancer cells using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA) was loaded and separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with the following antibodies overnight at indicated dilutions. pNFκB P65 ^S536, 1:1000, NFκB (P65), 1:1000, pGSK-3α^S21β^S9, 1:1000, pGSK3α^Y51β^Y47, 1:2000, GSK-3β, 1:1000, COX-2, 1:1000, pIKBα^S32, 1:1000, IKBα, 1:1000, pIKKα/IKKβ^S176/180, 1:1000, IKKα/IKKβ, 1:1000, pAkt^S473, 1:2000, Akt, 1:2000, β-Catenin, 1:1000, pβ-Catenin^S33/37/T41, 1:1000 (Cell Signaling Technology, Danvers, MA); Each membrane was probed with P84 (Gene Tex, Irvine, CA) to ensure consistent loading of nuclear protein and β-actin, 1:5000 (Sigma, St. Louis, MO) to ensure consistent loading of cytoplasmic protein.

Cells were lysed in NP-40 buffer (40 mM HEPES, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 8.0; 1% NP-40 (CA-630, Sigma); 5% glycerol; 10 mM pyrophosphate; 10 mM β-glycerophosphate; 50 mM NaF; 0.5 mM orthovanadate) containing Protease Inhibitor Cocktail (Sigma) and 1 mM DTT. Nuclear isolation was performed with a Nuclear Extract Kit (40010, Active Motif, Carlsbad, CA, USA), with 10 μg/ml ALLN (208719, Millipore, Bedford, MA, USA) treatment 20 min prior to isolation, and ALLN added to the hypotonic and lysis buffers. The nuclear fraction was washed with hypotonic buffer prior to lysis.
Antibodies used for immunoblots recognized SREBP1 (sc-8984, Santa Cruz, Santa Cruz, CA, USA), SREBP2 precursor and processed C-terminus (557037, BD, Franklin Lakes, NJ, USA), SREBP2 mature N-terminus (30682, Abcam, Cambridge, MA, USA), Actin (A5316, Sigma), and from Cell Signaling Technologies (Danvers, MA, USA): ACC1 (3676), FASN (3180), SCD (2438), HA (2367), P-Akt-T308 (9275), P-Akt-S473 (4051), Total-Akt (4691), P-S6K1-T389 (9234), Total-S6K1 (2708), P-S6-S240/S244 (2215), Total-S6 (2217), 4E-BP1 (9644), Ras (3965), P-Erk1/2-T202/Y204 (9106), Total-Erk1/2 (9102), Lamin A/C (2032), Histone H3 (4499).