Anti g6pd

Cells were lysed with lysis buffer (P89901; Thermo Scientific) containing protease inhibitors (P8340; Sigma-Aldrich). Western blot analysis was performed as previously described 10 (link). The following primary antibodies were used: anti-Trx-1 (1: 1000), anti-G6PD (1:2000), and GAPDH (1:5000) from Abcam (Cambridge, UK); anti-E-cadherin (1:1000; Cell Signaling Technology, Beverly, MA), and anti-vimentin (1:5000; Santa Cruz, CA, USA). Protein band intensity was quantified using Image Lab Software (Bio-Rad Laboratories Inc., Berkeley, CA, USA).

Each group of HCC samples was fixed in 10% formalin, embedded in paraffin, and processed as 5 μm continuous sections. Samples were dewaxed with discontinuous concentrations of ethanol and blocked to inhibit endogenous peroxidase. They were then heated in a microwave to retrieve antigens, cooled to room temperature, and then blocked by incubation in goat serum for 30 min at 37°C. Samples were incubated in rabbit anti-ANLN, anti-ENTPD2, anti-TRIP13, anti-PLAC8, anti-G6PD and anti-ADH1C (Abcam, Cambridge, UK; 1:1,200) overnight at 4°C, followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody at 37°C for 30 min and stained using 3,3′-diaminobenzidine. The cell nucleus was stained blue by hematoxylin. Sections were then dehydrated, cleared by xylene, and mounted. ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C expression were detected by IHC using a streptavidin peroxidase method. ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C expression in the liver were used as a positive control. Samples incubated with PBS instead of the ANLN, ENTPD2, TRIP13, PLAC8, G6PD, and ADH1C primary antibody were used as a negative control. Positive and negative controls were included for each batch of immunohistochemically stained sections. The experimental procedure was performed according to strict adherence to the manufacturers' instructions.

Unless otherwise stated, all chemicals were from Sigma-Aldrich Co., Ltd. Anti-Nrf2 (H300; sc-13032), Anti-Ub (sc-8017), anti-MKP-1 (sc-1102) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiserum against aldoketo reductases 1C1 and C2 (AKR1C) was kindly provided by Professor John Hayes (University of Dundee, Dundee, UK). Anti-IDH1 (Cat#: 3997) were from Cell Signaling Technology (Danvers, MA, USA). Anti-TKT (Cat#: ab88438), anti-ME1 (Cat#: ab87974), and anti-G6PD (Cat#: ab993) were from Abcam (Cambridge, United Kingdom). Actin antibody (Cat#: R1207) and IgG antibody (Cat#: HA1011) were purchased from Hua-an (Hangzhou, Zhejiang, China). Antibodies against NQO1, HO-1 or AKR1B10 were raised in this laboratory and described previously [37 (link), 38 (link)].

Equal amounts of extracts (20 μg) were separated by 10% SDS‒PAGE and transferred to PVDF membranes. After blocking with 5% milk in TBST, the following specific primary antibodies were used: anti-G6PD (1:1000; Abcam, UK), anti-VDAC1 (1:1000; Abcam), anti-Bax (1:2000; Abways, China), anti-Bcl-2 (1:500; Abways), anti-SM22α (1:2000; Abcam), anti-PCNA (1:1000; Abcam), anti-VDAC2 (1:1000; Abcam), anti-VDAC3 (1:500; Wanlei bio, China), anti-β-actin (1:1000; PTM bio, China), anti-α-actin (1:1000; PTM bio), anti-GAPDH (1:1000; Proteintech, China), anti-Tom40 (1:1000; Proteintech), anti-Caspase 9 (1:1000; PTM bio), anti-Cleaved Caspase 9 (1:1000; Wanlei bio), anti-Caspase 7 (1:1000; PTM bio), anti-cleaved Caspase 7 (1:1000; Wanlei bio), anti-Caspase 3/Cleaved Caspase 3 (1:1000; Wanlei bio), and anti-PARP/Cleaved PARP (1:1000; Wanlei bio), and incubated at 4 °C overnight. The blots were visualized using a GE ImageQuant™ LAS 4000 detection system (USA). Band intensities were quantified with ImageJ software (USA).

A Cell Counting Kit-8 (CCK-8, Cat# CK04) was purchased from Dojindo (Kumamoto, Japan). Pooled antisense oligonucleotides (i.e., siRNAs) against human OVOL2, P65, and GLUT1 were purchased from GenePharma (Shanghai, China). Antibodies against OVOL2 (Cat# ab101580) and P65 (Cat# ab16502) were purchased from Abcam (Cambridge, MA, USA). The following antibodies were used: Glycolysis Antibody Sampler Kits (Cell Signaling Technology; Cat# 12866 and Cat# 8337), anti-GLUT1, anti-GLUT2, anti-GLUT3, anti-GLUT4, anti-PGK1, anti-GCK, anti-PDP2, anti-DLD, anti-PCK1, anti-SDHA, anti-G6PD (all from Abcam; Cat# ab115730, ab234440, ab191071, ab188317, ab38007, ab184169, ab99170, ab133551, ab28455, ab14715, and ab210702, respectively), anti-GAPDH anti-Lamin B, anti-actin and anti-Calnexin (all from Cell Signaling Technology; Cat# 8884, 13435, 3700 and 2433, respectively).