Akt and p akt

Tumor tissues were mixed with RIPA Lysis buffer (Applygen Inc., Beijing, China) containing protease inhibitor cocktail (Roche, Switzerland). Lysates were centrifuged and supernatant was collected. After quantified using BCA protein assay kit (Pierce, Rockford, USA), 80 μg protein was separated by 6%-12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Pall, NY, USA) which was then blocked by 5% non-fat milk and incubated with primary antibodies: Bcl-2 (1:1000, Cell Signaling, Beverly, MA), Bax (1:1000, Cell Signaling), Caspase-3 (1:1000, Cell Signaling), AKT and pAKT (1:1000, Cell Signaling), VEGF (1:500, Abcam) at 4°C overnight, GAPDH (1:10000, sigma) at room temperature for 1hour, followed by horseradish peroxidase conjugated secondary antibodies (1:2000, Cell Signaling) incubation for another 1 hour at room temperature. The antigen-antibody complex bands were detected by enhanced chemiluminescence kit (ECL-Plus, Amersham Pharmacia Biotech, Piscataway, NJ, USA). GAPDH was used as loading control.

At sacrifice, 2–3 h after lights on, the serum was used for measurement of ghrelin and lactate, using a specific enzyme-linked immunosorbent assay kit^{15 (link)–20 (link)}. The low and high detection limits for ghrelin were 0.04 and 10 ng/mL (kit number EZRGRA-90K; Merck Millipore, Rosh Haayin, Israel), and the intra- and inter-assay CVs were 1.1% and 3.2%, respectively. Serum lactate was determined at the Soroka University Medical Center Biochemistry laboratory. In a subset of n = 4 of control animals, AO, and OR arterial blood gases (pH, PCO₂, PO₂, and HCO₃⁻) were determined in parallel to their serum lactate level on day 66 after surgery. Antibodies used for evaluation of the hypothalamic protein extract by western immunoblot analysis (see Supplementary Methods) were GHSR1a (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt and p-Akt (Cell Signaling Technology, Danvers, MA, USA), soleus AMPK and p-AMPK (MP Biomedical Solon, OH, USA), and GAPDH (Proteintech, Rosemont, IL).

Mice PFC were homogenized in protein lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1% protease inhibitor cocktail (Roche Molecular Biochemicals, Nutley, NJ, USA). Proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Blots were incubated in primary antibodies overnight at 4°C. The following primary antibodies were used: mature BDNF (Proteintech, 1:100), ERK and pERK (Cell Signaling Technology, 1:1000), AKT and pAKT (Cell Signaling Technology, 1:1000), GAPDH (Proteintech, 1:2000). The next day, blots were washed three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech, 1:3000) for one hour. The blots were detected by the enhanced chemiluminescence technique (Millipore, Billerica, MA, USA) and the results analyzed using Gel pro 3.2 (Media Cybernetics, Silver Spring, MD, USA). Protein levels were normalized to GAPDH levels and phosphoproteins were normalized to the total proteins and expressed as the percentage of the same protein found in control animals.

Total protein extract (30 μg of protein) or nuclear extract (10 μg of protein) from liver was resolved on an 8–10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA) and incubated for 2 hours in tris-buffered saline with Tween (TBST) (10 mM Tris, pH 8.0, 150 mM NaCL, and 0.1% Tween 20) containing 5% nonfat milk at room temperature. The membrane was then incubated overnight at 4°C with the following primary antibodies, SOD2 (1 : 5000, Epitomics), SOD1 (1 : 10000, Epitomics), CAT (1 : 1000, Epitomics), FOXO3a (1 : 1000, Epitomics), p-FOXO3a (ser253) (1 : 1000, Epitomics), HO-1 (1 : 2000, Epitomics), GCLC (1 : 5000, Bioworld), GCLM (1 : 1000, Epitomics), Nrf2 (1 : 500, Santa Cruz Biotechnology), p-Nrf2 (1 : 1000, Bioss), Akt and p-Akt (1 : 1000, Cell Signaling), Lamin B (1 : 500, Epitomics), and β-actin (1 : 500, Santa Cruz Biotechnology). After washing with TBST, the membrane was then incubated for 1 hour with horseradish peroxidase conjugated secondary antibody (all from Invitrogen with a dilution of 1 : 5000), and signal was detected using the SuperSignal West Pico Chemiluminescence kit on a Bio-Rad ChemiDocXRS Gel Imaging System. The band intensities were quantified by Quantity One Software version 4.6.3. Protein levels were normalized to β-actin or Lamin B as compared with the Control (set to 1).

MGC-803 cells were treated with various concentration of EAC-B for 24 h. Cell lysates were prepared in a buffer containing 0.2% (w/v) SDS, 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 1 mM PMSF, and 1% protease inhibitor cocktail. The total protein content was measured by BAC protein assay. The equal amount of protein was electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked, incubated overnight with primary antibodies at 4°C, and subsequently incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (Abcam). The following antibodies were used: Akt and p-Akt from Cell Signaling, cyclinD1, ERK, p-ERK, Bcl-2, Bax, GAPDH and β-actin from Santa Cruz. The blots were visualized using ECL detection reagents (Pierce).

Protein lysates were generated as described previously^{73 (link)} and separated using Criterion™ TGX™ Stain-Free™ gel (Bio-Rad Laboratories) followed by transfer onto nitrocellulose membrane (GE Healthcare). Equal sample loading was assayed by in-gel fluorescent detection or Ponceau S staining of the membrane. Membranes were blocked with TBS buffer containing 5% milk for 1 h and probed with specific antibodies. All antibodies used were purchased at Bethyl Laboratories with exception for: IGF1R, EIF3AK, pEIF3AK, ATF4, GNB1 and MYCN (Santa Cruz Biotechnology); AKT and pAKT (Cell signalling Technology); pEIF2S1 (Abcam); TUBB3 (BioLegend); AES (Novus Biologicals). Blots were scanned using ChemiDoc™ MP System (Bio-Rad) and bands were quantified Image Lab™ software (Bio-Rad). Uncropped blots of the main figures are shown in Supplementary Figure 8.