Anti p foxo1

Total protein was extracted from whole cells and 20 μg isolated protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was probed with anti-SOX9 (56KD) mouse monoclonal antibody (1:500; #ab76997, Abcam, Cambridge, MA, USA), anti-p-Akt(#4056), anti-Akt(#9272), anti-p21(#2947), anti-p27(#3686), anti-CyclinD1(#2922), anti-p-FOXO1(#9461), anti-FOXO1(#9454), anti-p-FOXO3(#9465) or anti-FOXO3(#2472) (Cell Signaling, Danvers, MA, USA). The membranes were then stripped and reprobed with an anti–α-tubulin mouse monoclonal antibody (1:1000; #2125; Cell Signaling, Danvers, MA, USA) as the loading control. Bound antibodies were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Dübendorf, Switzerland).

Anti-dystrophin (ab15277, 1:100 for immunostaining) and anti-VDAC1 (ab15895, 1:1000 for western blotting hereinafter) antibodies were purchased from abcam; anti-pY (4G10, 1:1000) antibody was from Upstate; and the others, anti-p-Akt (9271, 1:1000), anti-Akt (9272, 1:1000), anti-p-AMPKα (2531, 1:1000), anti-AMPKα (2532, 1:1000), anti-p-AS160 (4288, 1:1000), anti-AS160 (2447, 1:1000), anti-p-FoxO1 (9461, 1:1000), anti-FoxO1 (9454, 1:1000), anti-FoxO4 (9472, 1:1000), anti-GAPDH (2118, 1:5000), anti-LC3B (2775, 1:1000), anti-p-mTOR (2971, 1:1000), anti-mTOR (2972, 1:1000), antibodies were from Cell Signaling. Secondary antibody for immunostaining (Alexa 594 Goat Anti-Rabbit; A11037, 1:400) was purchased from Invitrogen, and those for western blotting (HRP-linked Anti-Mouse [from Sheep] and Anti-Rabbit [from donkey]; NA931 and NA934 respectively, both 1:2000) were from GE Healthcare Life Sciences.

Proteins from cell and tissue lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corp, Port Washington, NY). Then the membranes were blocked with 5% skimmed milk and incubated using primary antibodies against IMPDH2 (1:1000 dilution), anti-GAPDH, anti-GSK3β, anti-p-GSK3β, anti-AKT, anti-p-AKT (Ser473), anti-FOXO1, anti-p-FOXO1, anti-mTOR, anti-p-mTOR (Cell signaling Technology, Beverly, MA), E-cadherin (1:1000 dilution,#SAB4503751; Sigma Aldrich), β-catenin (1:1000 dilution, #C2206; Sigma Aldrich), Vimentin (1:1000 dilution, #V6630; Sigma Aldrich), Snail (1:1000 dilution, #SAB1306281; Sigma Aldrich), followed by incubation with the appropriate secondary antibodies (anti-rabbit IgG, 1:3000 dilution, #7074; Cell Signaling). An enhanced chemiluminescence (Pierce, Rockford, IL, USA) was used to detect signals.

Tissues were dissolved in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, protease and phosphatase inhibitor mixture (Roche Diagnostics)). Protein concentrations were determined using a BCA assay kit (Pierce Diagnostics). Protein was separated by 10% (wt/vol) SDS/PAGE, transferred to a PVDF membrane (Millipore), blocked in 5% (wt/vol) skim milk in TBST (0.02 M Trisbase, 0.14 M Vehicle, 0.1% Tween 20, pH 7.4), and incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with HRP. The following primary antibodies were used: anti-UCP1 (ab10983, Abcam), anti-PGC1ɑ (ab54481, Abcam), anti-OXPHOS (ab110413, Abcam), anti-Mas1 (AAR-013, Alomone labs), anti-Akt (#9272, cell signaling technology), anti-p-Akt308 (#13038, cell signaling technology), anti-FoxO1 (#2880, cell signaling technology), anti-p-FoxO1 (#84192, cell signaling technology), anti-PKA (#4782, cell signaling technology), anti-p-PKA (#9621, cell signaling technology), anti-ACE2 (#92485, cell signaling technology), and actin (#4970, Cell Signaling Technology). Signals were detected with Super Signal West Pico Chemiluminescent Substrate (Pierce).

B cells cultured under various conditions were harvested and cell lysates were prepared in lysis buffer. The lysate was resolved on a 10% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with antibodies, followed by probing with HRP-conjugated anti-rabbit or -mouse antibody (Sigma). The bands were detected by chemiluminescence with ECL detection reagents (Life Technologies). Antibody to Phospho-STAT6 (Tyr641, 56,554), anti-STAT6 (5,397), anti-p-PKA C(4,781), anti-PKA C-α (5,842), anti-p-CREB (9,198), anti-CREB (9,197), PI3 Kinase p110 δ 34,050, anti-p-Akt (Ser473, 4,060), anti-Akt (4,685), anti-p-FoxO1 (Ser256, 9,461), anti-FoxO1 (2,880), anti-p-FoxO3a (Ser253, 13,129), anti-FoxO3a (12,829), anti-PPARγ (2,443) were from Cell Signaling Technology (Danvers, MA, USA). Anti-ubiquitin (PTM-1106) was from PTM BIO (Beijing, China). Anti-β-Actin (66009-1-Ig) was from Proteintech.

Cells were lysed in RIPA buffer, supplemented with SigmaFast^TM protease and phosphatase inhibitors (Sigma Aldrich; UK), and protein concentration was determined by the Pierce BCA Protein Assay (ThermoFisher Scientific; UK). Protein samples (30 µg) were separated by SDS-PAGE at 150 V on 10% Tris-Glycine mini gels (ThermoFisher Scientific, UK) for 60–90 min. Resolved proteins were transferred to Hybond^TM polyvinylidene difluoride (PVDF) membrane (0.45 µm; GE Healthcare; UK) by semi-dry transfer (0.8 mAmps/cm²). Membranes were blocked (5% milk or 2.5% BSA; 1 h) and probed with primary antibodies at 4 °C overnight (anti-β-actin 1:5000 (Sigma Aldrich; UK); anti-FOXO1 1:1000; anti-pFOXO1 1:500; anti-CPT1A 1:1000 (Cell Signaling Technology; UK)) followed by incubation with HRP-conjugated secondary antibodies (1:10,000 in 0.2% BSA; 1 h (ThermoFisher Scientific; UK). Proteins were detected by enhanced chemiluminescence (ECL) using Western Lightning plus ECL reagent (Perkin-Elmer; UK). Band densities were semi-quantified by densitometry performed using ImageJ software and normalised to β-actin loading control.