Luminol reagent

The cell lysate from each group was prepared using a RIPA lysis buffer containing protease inhibitor. Samples were centrifuged and the total protein concentration of supernatants was determined by Lowry protein assay. Twenty μg of protein was mixed with an equal volume of sample buffer and electrophoresed on SDS–PAGE, then transferred onto PVDF membranes (Santa Cruz, USA). After blocking with 5% skimmed milk, the membrane was washed in PBS with Tween detergent and treated with primary antibodies against the pluripotency markers Oct4 (Abcam, USA) and Sox2 (Abcam, UK), endodermic markers CXCR4 (Santa Cruz, USA), and Sox17 (Santa Cruz, USA), and the internal control β-actin (Santa Cruz Biotechnology, USA) overnight at 4 °C. Primary antibodies against Wnt3a (Santa Cruz, USA) and β-catenin (Santa Cruz, USA) were applied for evaluation of the Wnt signaling pathway. Then, the membrane was exposed to horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. Finally, the membrane was visualized by luminol reagents (Santa Cruz, USA). The intensity of the bands was quantified using ImageJ software (version 1.41).

For Western blot (WB) analysis, equal amounts of protein (50 µg) in RIPA buffer from each cell type were resolved by 12% SDS-PAGE under reducing conditions. Gels were transferred to nitrocellulose membranes (Bio Rad, Hercules, CA, USA), blocked with 5% non-fat milk, washed with PBS 0.05% Tween 20 (v/v) (PBS-T), and then incubated for 1 h at RT with a 1:1000 dilution of purified rabbit IgG to Myo1g as a positive control and a 1:4000 dilution of supernatant (SN) from the 3 mAb to Myo1g in PBS-T, or a 1:5000 dilution for anti-actin mAb (Sigma-Aldrich, St Louis, MO, USA). Subsequently, they were washed 3 times with PBS-T, and incubated for 1 h at RT with a 1:15,000 dilution of HRP-coupled secondary Ab GAR (Abcam, Cambridge, UK), 1:20,000 of GAM (Abcam, Cambridge, UK). After 3 further washes with PBS-T, luminol reagents were added (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The chemiluminescence analysis was performed on a Fusion FX-7 Spectral Imaging System (Vilber, Paris, France).

Western blot analysis (WB) was performed to analyze protein expression and activation after cells were treated with Olanzapine (40 μM), Risperidone (40 μM), Amisulpride (30 μM) and Aripiprazole (10 μM). Briefly, cells were washed in PBS, collected and then lysed in RIPA buffer (radio immunoprecipitation assay buffer, 50 mM Tris-HCl; 150 mM NaCl; 0.1% SDS; 0.5% Na-deoxycholate; 1% NP40) containing proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). The lysate was centrifuged at 12000 rpm at 4 °C for 15 min, and equal protein lysate was used for Western blot analyses. For WB, 50 μg of total cell lysate was subjected to SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane (Pall Corp, Ann Arbor, MI), and the membranes were blocked in 1 × PBS, 0.1% Tween-20 and 5% skim milk. After blocking, the membranes were incubated with primary antibodies diluted 1:1000 in 1 × PBS, 5% skim milk and 0.1% Tween-20 overnight at 4 °C. Immunodetection was performed by the Western blotting Luminol Reagent (Santa Cruz Biotechnology Inc. CA, USA). Actin immunoblotting was performed to verify that equal protein had been loaded in each lane. Their optical density was analyzed with Quantity One software. The expressions of target proteins were normalized to β-actin.

Whole cell lysates were prepared using TNTE lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA and 0.3% Triton-X100, supplemented with 1× Complete protease inhibitor cocktail (Roche) and 1× PhosSTOP (Roche)]. Lysates were cleared by centrifugation and 4× SDS-Sample buffer was added to the clear supernatant. Samples were boiled for 5 min at 95°C. Proteins were loaded in equal amounts and resolved on Tris-Glycine 4–12% gels (Bio-Rad), following transfer to a PVDF membrane (Millipore). Following incubations with primary and secondary antibodies, the blots were developed using Luminol reagent (Santa Cruz). Mouse antibodies used in this study: anti-WIPI2 (2A2 clone Abcam, ab105459) and Vinculin (Sigma, V9264). Rabbit antibodies used in this study: anti-p62 (Enzo Life Sciences, PW9860) and anti-LC3B (Abcam, ab48394). Densitometry was performed with ImageJ software.

40 μg of heat-denatured proteins were loaded on 4–15% precast polyacrylamide gel (Bio-Rad). The proteins were then transferred to PVDF membranes (Bio-Rad), which were blocked with 5% non-fat milk solutions for 1 hour at room temperature. The target proteins were then detected by the primary antibody at 4 °C overnight, washed with 0.1% Tween-PBS and incubated with appropriate secondary antibody for 2 hours. The membranes were then washed and the target proteins were detected with luminol reagent and X-ray film (Santa Cruz). Quantification of the target protein was done using Adobe Photoshop. In brief, the background of the target protein and β-actin were subtracted. Then, the relative expression of the target protein was normalized to β-actin and compared to that of the control group in each experiment. Anti-human HIF-1α antibody was from Novus-bio. Rabbit anti-human BIRC3, goat anti-Rabbit IgG-HRP and anti-β-actin IgG-HRP were obtained from Santa Cruz Biotech.

Protein was isolated from samples using Trizol (Invitrogen). Protein concentration was measured using the BCA kit (Thermo Scientific) and 40 μg of protein/lane loaded in a 10% SDS PAGE precast gel (Invitrogen). Gel was transferred using the iBlot transfer system (Invitrogen). Nitrocellulose membrane was blocked in 2% I-Block (Applied Biosystems) in 1×TBS-T for one hour, and then either b-actin (Abcam, 1:10,000), Histone H3 (Cell Signaling, 1:1000), RAGE (R&D Biosystems, 1:500), TLR4 (Santa Cruz, 1:500), or NFκB (Abcam, 1:1,000) was used. Secondary antibodies (anti-mouse, Jackson ImmunoResearch) were added at 1:1,000 dilution in 2% I-Block in 1×TBS-T on a room temperature. The membranes were washed with 1×TBS-T, and then Luminol Reagent (Santa Cruz) was added. The membranes were then developed using a FluorChem E Imager system (ProteinSimple).