Supersignal west pico chemiluminescent substrate system

Samples were prepared in radioimmunoprecipitation assay (RIPA) buffer, boiled, and separated by SDS-PAGE on 10% polyacrylamide gels, followed by transfer to Immobilon-P membranes (Millipore) using the Trans-Blot semi-dry electrophoretic transfer system (Bio-Rad). Membranes were blocked for 1 h at RT with 5% skim milk in TBS containing 0.1% Tween 20 (TBST) and then incubated at 4°C overnight with primary antibodies specific for α-SMA (Sigma-Aldrich), tubulin (Cell Signaling), phospho-Smad2 (Cell Signaling), phospho-Smad3 (Abcam), Smad2 (Cell Signaling), Smad3 (Abcam), integrin beta 3 (Abcam), integrin beta 5 (Abcam), integrin beta 6 (R&D systems), and β-actin (Cell Signaling) (all diluted in 2% skim milk in TBST). Membranes were washed and then incubated with HRP-conjugated goat anti-mouse, goat anti-rabbit, or donkey anti-goat IgG secondary antibodies (Jackson ImmunoResearch, Suffolk, UK) diluted in 2% skim milk in TBST. Blots were developed with the SuperSignal West Pico chemiluminescent substrate system (Thermo Fisher Scientific).

For immunoblot analysis, whole-cell lysates were obtained using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with Complete inhibitor cocktail tablet (Roche, Germany). Protein concentrations were quantified using the RC DC protein assay (Bio-Rad, USA). Protein samples were subjected to SDS-PAGE (4–15% gradient gels, Biorad, USA) and transferred to nitrocellulose using Trans-Blot® TurboTM transfer system (Bio-Rad, USA). After blocking for 1 hour at room temperature (RT) in TBS containing 0.1% Tween and 2% milk powder, membranes were incubated overnight with primary antibodies at 4 °C. Primary antibodies used in this study are: anti-Glut5 (Millipore cat# 07-1406, 1:1000) and anti-GAPDH (Calbiochem, Darmstadt, 1:5000). Antibodies were detected by incubating the blot with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit (Life science, NA934, 1:2500) or rabbit anti-mouse (Dako, p0260, 1:5000) IgG for 1 hour at RT. Protein bands were detected using ChemiDoc (Bio-Rad, USA) after incubation with the SuperSignal West Pico Chemiluminescent Substrate System (Thermo Fisher Scientific, USA). GLUT5 levels in samples were calculated relative to GAPDH using Image lab 3.0 software (Bio-Rad Laboratories, USA).

Cells were lysed with RIPA (50 mM Tris-HCl pH 7.4, 150 nM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate and 1 mM EDTA) or with 2x denaturing loading buffer. Samples were heated at 95° C during 5 min and separated on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and blocked with 10% (w/v) semi skimmed dried milk. Blocked membranes were incubated overnight with primary antibodies (anti-HIF-1α 1:500, anti-PHGDH 1:1000, anti-α-tubulin 1:10000, anti-calnexin 1:1000), washed and incubated with the peroxidase-linked secondary anti-rabbit or anti-mouse antibody for 1 h at room temperature. Membranes were washed and finally incubated with the Supersignal^® West Pico chemiluminescent substrate system (Thermo Scientific). Image captions were taken with the ChemiDoc^TM XRS+ System (Bio-Rad) using Image Lab software or either films were revealed using a Medical film processor from Konica Minolta (Tokyo, Japan). Densitometry analyses were made with ImageJ software.

For immunoblotting, kidney lysates were prepared as previously described [39 (link)]. Proteins were detected with specific antibodies against Stat1 (Cell Signaling), phospho-Stat1 (Cell Signaling), β-Actin (Sigma) and active β-Catenin (Millipore). Blots were developed using a peroxidase-conjugated secondary antibody (Sigma) and an enhanced SuperSignal West Pico Chemiluminescent Substrate system (ThermoFisher Scientific) according to manufacturer's instructions. For ELISA, lysates were prepared from kidneys at E14.5. The amount of the Ifng was measured with a mouse Ifng Femto-High Sensitivity ELISA kit (eBioscience).

Liver samples (∼100 mg) from snap-frozen mouse liver tissue were homogenized using 1 ml of buffer A [1% SDS, 1% Triton X-100%, 1% NP-40, 50 mM Tris-HCl (pH 7.4), 50 mM EDTA] containing a mixture of protease inhibitors (2 mM PMSF, 50 μg/ml ALLN, 20 μg/ml aprotinin, 50 μg/ml leupeptin, and one tablet of Roche protease inhibitor per 10 ml). Equal aliquots of protein from individual livers within each group were pooled (total, 40 μg), and proteins were subjected to 8% SDS-PAGE gels and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). Immunoblot analyses were performed using SREBP-1, SREBP-2, FAS, acetyl-CoA carboxylase (ACC)1, ACC2, ATP citrate lyase (ACL), insulin receptor substrate (IRS)1, phospho-IRS1, IRS2, phospho-IRS2, protein kinase B (AKT), phospho-AKT(Ser473), phospho-AKT(Thr308), mammalian target of rapamycin (mTOR), phospho-mTOR, S6 ribosomal protein, and phosphor-S6 ribosomal protein antibodies listed in supplemental Table S1. SuperSignal West Pico chemiluminescent substrate system (Thermo Scientific) was used to detect the antibody-bound bands. Anti-CREB and anti-β-actin antibodies were used as loading controls for the proteins, respectively.