Hrp conjugated secondary antibody

The UB/OC-2 cells were purchased from Ximbio (London, UK). Gentamicin was obtained from Standard Chem & Pharm Co., Ltd (Tainan, Taiwan). THSG was purchased from MedChemExpress Company (Monmouth Junction, NJ, USA). MTT was obtained from VWR International (Radnor, PA, USA). LDH cytotoxicity assay kit, DCFDA, Hoechst 33258 dye, JC-1 dye, Annexin V, and PI were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Primary antibodies targeting PARP, cytochrome c, β-actin, COX IV, cleaved caspase 9, cleaved caspase 3 were purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-conjugated secondary antibodies were obtained from PerkinElmer Life Sciences (Boston, MA, USA). SOD activity assay kit and the cell fractionation kit were purchased from Abcam (Cambridge, MA, USA).

HEK-293T cells, transiently transfected with SARS-CoV-2 plasmids or with empty control were collected, washed in ice-cold phosphate-buffered saline and subsequently lysed in ice-cold 50 mM Tris-HCl (pH 7.4), 0.25% sodium deoxycholate, 1% Nonidet P-40, 150 mM sodium chloride, 1mM sodium orthovanadate, 1mM sodium fluoride, 2.5 mM sodium pyrophosphate, 1mM EDTA, 1mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail (cat #539,134, Calbiochem, Merck KGaA, Darmstadt, Germany). Whole cell lysates were quantified using the Pierce BCA protein assay kit (cat. 23,227, ThermoFisher, Waltham, MA, USA) and 25 μg of total proteins were incubated at 95 °C for 10 min, loaded on SDS-PAGE gels and then transferred to Hybond-ECL membranes (Amersham, Biosciences Corp, Amersham, UK). The membranes were blocked with 5% milk/0.3% TBS-Tween 20 at 4 °C for 1 h and then probed with primary antibodies against GFP (1:1,000 dilution; cat. ab290, AbCam, Cambridge, UK) or Strep-tag II (1:1000 dilution; cat. ab184224, AbCam, Cambridge, UK) and β-Actin (1:6000 dilution; cat. A1978, Sigma-Aldrich, Darmstadt, Germany). HRP-conjugated secondary antibodies (Cat. NEF812001EA, Perkin Elmer, Waltham, MA, USA) were used at 1:10,000 dilution. The chemiluminescent signal was detected with enhanced chemiluminescence (ECL) (cat.32,106, ThermoFisher, Waltham, MA, USA) and subsequently autoradiography.

Tissue samples were homogenized and lysed in RIPA (Bio-Rad Laboratories, Hercules, CA, USA) buffer, run on a 12% polyacrylamide gel, and wet transferred to Bio-Rad PVDF membrane (Bio-Rad Laboratories) according to the standard protocol and chemiluminescently visualized using HRP-conjugated secondary antibodies from PerkinElmer (Waltham, MA, USA). Anti-GAS2 (ab55076-100) from Abcam (Cambridge, MA, USA) and anti-laminin (L9393) from Sigma-Aldrich (St. Louis, MO, USA).

Cell pellets were lysed with RIPA buffer (Sigma-Aldrich) following the manufacturer’s instruction. SDS–polyacrylamide gel electrophoresis was performed using 4–20% Criterion TGX Stain Free Protein Gel (Bio-Rad, Hercules, CA), and proteins were transferred to poly-vinylidene difluoride (PVDF) Immobilion-p membrane (Merck-Millipore, Billerica, MA, USA) using wet/tank Bio-Rad blotting system. Membranes were blocked in I-block 2% (Invitrogen, Waltham, MA, USA), incubated overnight at 4 °C with primary antibodies and 1 h with the HRP- conjugated secondary antibody (PerkinElmer, Waltham, MA, USA). Bands were detected using Invitrogen™ iBright™ FL1500 Imaging System. The following primary antibodies were used: JAK1 Y1022/1023 (Cell Signaling Technologies, #3331), JAK2 Y1007 (MBS128333, MyBioSource, San Diego, CA, USA), STAT6 Y641 (Millipore, 06-937), STAT3 Y705 (Cell Signaling Technologies, #9145), STAT5 Y694 (Cell Signaling Technologies, #9351), STAT6 (#11290, Becton Dickinson, Franklin Lakes, NJ, USA), STAT3 (79D7) (Cell Signaling Technologies, #4904), STAT5 (Becton Dickinson #611834), GAPDH (GTX 8627408, Genetex, Irvine, CA, USA).