Biospectrum image system

Cells (5×10⁵ or 1×10⁶) were lysed in protein lysis buffer (2% SDS, 50 mM Tris-HCl, pH 7.5) containing protease inhibitor and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) 26 (link). Protein concentration was determined by using a Bradford assay kit (BioRad, Hercules, CA, USA). An amount of 50-80 µg protein lysates were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes, which were blocked with 5% milk in Tris-buffered saline (TBS) for 1 h at room temperature, and then incubated with primary antibody overnight at 4 °C. After washing with TBST buffer (TBS with 0.05% Tween X100), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 2 h at room temperature and then revealed using enhanced chemiluminescent (ECL) reagent (Advansta, San Jose, CA, USA). Image and emission signal density measurements were quantified by using a BioSpectrum Image System (UVP, Upland, CA).

Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40,
0.1% SDS, 50 mM Tris-HCl [pH 8.0]) containing protease inhibitor (Roche).
Harvested cell extracts were separated by 10% SDS-PAGE and transferred to PVDF
membranes, which were incubated with primary antibody, then horseradish
peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory) and
visualized by an enhanced chemiluminescence system (Thermo). Images were
acquired by use of BioSpectrum Image System (UVP, Upland, CA).

Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) or SDS buffer (2% SDS, 50 mM Tris-HCl [pH 7.5]) containing protease inhibitor and phosphatase inhibitor cocktail (Roche). Harvested extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes. Later, the membranes were incubated with primary antibody, and then with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and were finally visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Image acquisition and signal density measurements were carried out using the BioSpectrum Image System (UVP, Upland, CA). The following primary antibodies were used: anti-caspase 1 (GTX111630, GeneTex, Irvine, CA), anti-caspase 3 (#9665, Cell Signaling Technology, Danvers, MA), anti-caspase 7 (GTX1002337, GeneTex), anti-caspase 8 (#4790, Cell Signaling Technology), anti-bone morphogenetic protein 4 (BMP-4; GTX100875, GeneTex), anti-phospho-STAT1 (phospho-Tyr701, #9167 Cell signaling), anti-STAT1 (#14994, Cell Signaling), anti-phospho-STAT2 (phospho-Tyr690, GTX50721, GeneTex), anti-STAT2 (#14994, Cell Signaling), anti-DENV NS3 (GTX124252, GeneTex), and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL).