Irdye fluor 800 labeled igg

Whole-cell lysates were prepared in RIPA lysis buffer and measured for protein concentration by BCA protein assay kit (Life Technologies, USA) to equalize protein loading. Proteins were resolved in SDS-PAGE gels and transferred to a PVDF (polyvinylidene difluoride, Millipore, USA) membrane at 100 V for 60 min (Bio-Rad). All membranes were blocked in 3% BSA in TBS with 0.5% Tween 20 (TBST; pH 8.0; Sigma) and probed with indicated antibodies in antibody dilution reagent solution (Life Technolies, MD, USA) at 4°C overnight, and then incubated with either IRDye Fluor 800-labeled IgG (1:10000 926–32210) or IRDye Fluor 680-labeled IgG (1:10000 926–68071) secondary antibody (Li-COR Bioscience). The images were scanned and quantified by densitometric analysis on Li-COR Odyssey Infrared Imager. Primary antibodies includes antibodies specific for caspase 3, caspase 8, Bcl-2, BAX, and GAPDH (Proteintech), antibodies specific for phospho-VE-cadherin (Tyr685; Cell Signaling) and antibodies specific for VE-cadherin (CST). Rabbit anti-SFTSV-Gn antibodies were kindly provided by Y-Clone, Ltd.

Cell lysates were prepared in standard RIPA buffer (Santa Cruz, USA) and cleared by centrifugation. Total proteins were quantified by BCA protein assay kit (Life Technologies, USA) and separated on 10% SDS-polyacrylamide gel, and transferred onto PVDF membranes (Millipore, USA). Proteins were detected with respective antibodies at 4°C overnight, followed by incubation with either IRDye Fluor 680-labeled IgG (1:10000 926-68071) or IRDye Fluor 800-labeled IgG (1:10000 926-32210) secondary antibody (Li-COR Bioscience). The images were scanned and quantified by densitometric analysis on Li-COR Odyssey Infrared Imager. Rabbit anti-STAT1 (1:1000 10144-2-AP) and STAT3 (1:1000 10253-2-AP) and mouse anti-GAPDH (1:1000 60004-1-Ig) antibodies were from Proteintech (China). Rabbit anti-phospho-STAT1 (Ser727) mAb (1:1000 D3B7) was purchased from Cell Signaling Technology, USA. Rabbit anti-SFTSV NP antibody (1:500 01-05-0130) was from Cambridge Bio (UK). Rabbit anti-SFTSV NSs antibody (1:1000 E7914) was custom ordered from ABclonal (China).

To collect proteins, cell samples or homogenized skin tissues were lysed with RIPA lysis buffer (Santa Cruz, USA). After being centrifuged at 12,000×g for 10 min at 4°C, the cleared lysate was collected, and a BCA protein assay kit (Pierce, Rockford, IL, USA) was used to determine the total protein concentrations. The collected protein was subjected to 10% SDS-polyacrylamide gel for protein separation and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked, followed by incubation with respective primary antibodies at 4°C overnight. After washing with PBST (0.1% Tween-20), the membranes were incubated with corresponding secondary antibody (IRDye Fluor 680-labeled IgG or IRDye Fluor 800-labeled IgG (LI-COR, Bioscience)) for 1 h at room temperature and visualized using LI-COR Odyssey Infrared Imager (LI-COR, Bioscience).