Anti β actin gtx109639

Oleanolic acid was isolated from benzoin and was prepared by Shenzhen University. Oleanolic acid was dissolved in dimethyl sulphoxide with a concentration of 50 mM. Acyclovir (ACV) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies, including anti-gB (ab6506), anti-ICP0 (ab6513), anti-gD (ab6507), anti-VP16 (ab11026), anti-ICP4 (ab6514), anti-ICP8 (ab20194), anti-ICP27 (ab53480), anti-ICP22 (ab6506), were purchased from Abcam (Cambridge, United Kingdom), anti-β-actin (GTX109639) were purchased from GeneTex, anti-GAPDH (2118) were obtained from Cell Signaling Technology (Danvers, MA, United States). All the eukaryotic expression plasmids, including p3Xflag-UL5, pCMV-HA-UL8, pEGFPC1-UL52, were generated in our laboratory. Additional information about plasmids construction and primer sequences was shown in Supplementary Table 1. All siRNAs were purchased from Gene Pharma (Shanghai, China), and their sequences were shown in Supplementary Table 2.

Cells were lysed in RIPA Buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing a protease and phosphatase inhibitor cocktail (Fujifilm Wako Pure Chemical Corporation, Kyoto, Japan). Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Ten micrograms of protein were denatured in Laemmli sample buffer and loaded on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Richmond, CA, USA). After electrophoretic separation, the proteins were transferred onto PVDF membranes (MilliporeSigma, Billerica, MA, USA). Membranes were blocked overnight at 4 °C with Tris-buffered saline (TBS) containing 5% non-fat dried milk or bovine serum albumin. Following blocking, membranes were incubated overnight at 4 °C with primary antibodies, including anti-HIF-1α (ab179483; 1:1000; Abcam, Cambridge, UK), anti-HIF-2α (ab109616; 1:1000; Abcam), and anti-β-actin (GTX109639; 1:1000; GeneTex, Irvine, CA, USA). Proteins were incubated with secondary antibodies (anti-rabbit or anti-mouse IgG antibody; Cat. 7074 and Cat. 7076, respectively; 1:20,000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in TBS containing 5% non-fat dried milk. The chemiluminescence signal was detected using enhanced chemiluminescent substrate (SuperSignal™ West Femto Trial Kit; Thermo Fisher Scientific).

Anti-GSTM1 (GTX113448 for mouse, GTX100298 for human), anti-β-actin (GTX109639) and anti-α-tubulin (GTX112141) antibodies were purchased from GeneTex (Taichung, Taiwan). Anti-NAD(P)H quinone oxidoreductase-1 (NQO1) (ab2346) was purchased from Abcam (Cambridge, UK). Antibodies against p21 (sc-397) and nuclear factor erythroid 2-related factor 2 (Nrf2) (sc-722) were purchased from Santa Cruz (Dallas, Texas, USA). Peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).