Anti akt

SCC-9 and SCC-14 cells were seeded 6 × 10⁵ and 1.2 × 10⁶ onto 6 cm dish, respectively, and treated with sulforaphane. Cell lysate were collected with 50–100 μL of protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) as previously described [48 (link)]. After centrifuged at 13000 g at 4° C for 30 min. The protein lysate were separated by 12% agarose gel and transferred onto a nitrocellulose membrane then blocking with 5% non-fat milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h in room temperature and overnight with first-antibodies in 4° C and second-antibodies for 1 h in room temperature. Anti-p-ERK, anti-ERK, anti-JNK, anti-p-JNK, anti-p-Akt, anti-LC3I, anti-Beclin 1, anti-p-mTOR, anti-mTOR were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Akt, anti-pp38, anti-p38 were purchased from BD Biosciences (Bedford, MA, USA). Anti-cathepsin S was purchased from GeneTex (CA, USA). The band intensities were quantified by densitometry.

For Western-blotting, 50 μg of total protein from cell lysates was boiled and resolved by 6%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), depending on the molecular weight of the proteins to be analyzed. After electrophoresis, proteins in gels were transferred to polyvinylidene difluoride membranes (Millipore Corporation). Blots were blocked in 1x Tris-buffered saline (TBS,100 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20) and 1% of bovine serum albumin for 1 hour and then incubated overnight with the following primary human monoclonal/policlonal antibodies: anti-AKT, anti-p^S473-AKT, anti-pH2AX, anti-pCHK2 (BD Biosciences), anti-ERK1/2, anti-pERK1/2, anti-PARP-1 (Santa Cruz Biotechnology), anti-pS6, anti-p^T308-AKT (Cell Signalling Technology), anti-Src (Cell Signalling Technology). Protein-bound primary antibodies were detected using respective horseradish peroxidase-coupled secondary antibodies (anti-rabbit for polyclonal and anti-mouse for monoclonal, obtained from Santa Cruz Biotechnology) diluted 1:5,000 in 1x TBS containing 0.05% Tween and incubated for 1 hour at room temperature. Protein bands were detected using ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, United Kingdom).

Cells plated in media containing 5% FCS were treated with solvent (0.1% DMSO) in the absence or presence of various concentrations of FASN-inhibitor for 1 to 72 h. After lysis, proteins were subjected to SDS–PAGE, blotted and immunostained as described [45 (link), 46 (link)] using anti-FASN (BD Biosciences, San Jose, CA; 1:500), anti-AKT, anti-pAKT, anti-AMPKα, anti-pAMPKα, anti-DEPTOR, anti-ERK, anti-pERK, anti-HIF-1α, anti-mTOR, anti-pmTOR, anti-p70S6K, anti-pp70S6K, anti-S6, anti-pS6, anti-4EBP1, anti-p4EBP1, anti-cleaved caspase 3, anti-PARP1 (Cell Signaling Technology, Boston, MA; 1 : 500–1:3 000), anti-REDD1 (Proteintech, Manchester, UK; 1:1 000), and anti-actin (Santa Cruz Biotechnology, Dallas, TX; 1:1 000). Secondary antibodies were peroxidase-labelled donkey-anti-rabbit (Promega, Madison, WI) or donkey-anti-goat IgG (Santa Cruz Biotechnology) at 1:15 000, or chicken-anti-mouse IgG (Santa Cruz Biotechnology) at 1:10 000. Detection was by enhanced chemiluminescence.

Antibodies against phospho-AKT(Ser473), ERK, phospho-ERK(Thr202/Tyr204), STAT5, and phospho-STAT5 were obtained from Cell Signaling Technology (Danvers, MA); anti-KIT from R&D Systems (Minneapolis, MN) and Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-KIT(Tyr823) from Invitrogen (Carlsbad, CA); anti-AKT from BD Biosciences (San Diego, CA); and anti-β actin from Sigma Aldrich (St. Louis, MO); and anti-rabbit IgG 800CW and anti-mouse IgG 680 RD from Licor Biosciences (Lincoln, NE). Human TruStain FcX and APC-conjugated anti-human c-KIT (clone 104D2), unlabeled anti-human KIT antibody (clone 104D2) and Zombie Aqua fixable viability dye were obtained from Biolegend (San Diego, CA); KIT antibody for IHC from Dako-Agilent (Santa Clara, CA); human SCF from R & D Systems; avapritinib, imatinib, dasatinib, midostaurin, fedratinib, STAT5-IN-1, SH4-54, venetoclax, ripretinib (DCC-2618), ruxolitinib, tofacitinib, and C188-9 from SelleckChem (Houston, TX); LY294002 and U0126 from Tocris (Minneapolis, MN); Pierce BCA assay and CyQuant cell proliferation assay from Thermo Fisher Scientific (Waltham, MA); and ViaStain propidium iodide (PI) staining solution from Nexelom Biosciences (Lawrence, MA).

Lysis of cells and immunoprecipitation were carried out as described previously^{10 (link)} using anti-BTG3 or anti-AKT (no. 610860; BD Biosciences).

After 2 days, the cells were washed with PBS and lysed in lysis buffer containing 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, and protease inhibitors (Millipore, Bedford, MA) and subsequently incubated on ice for 30 min. The mixtures were then centrifuged (13,000 rpm; 30 min). The protein concentrations were measured with a BCA kit. The protein extracts were electrophoresed on 6% Bis-Tris protein gels and then transferred to PVDF membranes. After incubated in 5% dry milk for 1 h, the membranes were incubated with the following primary antibodies: anti-p-AKT, anti-AKT (BD Biosciences), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bioworld, USA). Then, the membranes were incubated with the peroxidase AffiniPure goat anti-rabbit IgG (H + L chains; Jackson ImmunoResearch Laboratories, West Grove, PA). The enhanced chemiluminescence (ECL) kit (Amersham, UK) was used to detect the protein bands. The Image-Pro Plus image analysis software was used to quantify the band intensity. The mean grey value was used for further statistical analysis.