PI3K inhibitor – Western blot images
Description
Data in the paper "Cellular state landscape and herpes simplex virus type 1 infection progression are connected" by Pietilä et al., 2023: - Images of Western blots (Supplementary Figure 13) - Fold changes quantified from Western blots (Figure 6b) Three independent experiments were performed to analyse how a PI3K inhibitor (LY294002) affects HSV-1-induced Akt phosphorylation in HeLa cells. DMSO treatment was used as a control. Band intensities of HSV-1 ICP5, ICP8, and ICP27 as well as of p-Akt (Thr308), p-Akt (Ser473), and beta-actin were quantified, and results are shown in Figure 5f of the paper. A summary of the Western blots is shown in Figure S8e of the paper.
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30,000 HeLa cells were seeded per well in 12-well plates and grown at 37°C and 5% CO2 for ~48 h. Cells were infected with HSV-1 diluted in serum-free DMEM using MOI 1. Cells were then incubated with the virus for 30 min at 4°C, and then unbound virus was removed by washing cells with warm DMEM supplemented with 10% (v/v) FBS. Cells were subsequently grown at 37°C. At 1.5 hpi, cells were treated with 0.1% (v/v) DMSO or 50 µM LY294002 (containing 0.1% (v/v) DMSO). At 12 hpi, cells were washed with ice-cold PBS and collected in 2× Laemmli sample buffer (4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol, 0.1% (w/v) bromophenol blue, 100 mM Tris-HCl pH 6.8) and incubated for 5 min at 99°C. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 4% (w/v) acrylamide in the stacking gel and 10 or 12% (w/v) acrylamide in the separation gel. After electrophoresis, proteins were transferred to nitrocellulose blotting membrane, which were subsequently blocked with 5% (w/v) milk in PBS and incubated with primary antibodies. Subsequently, membranes were washed with 0.3% (v/v) Tween 20 in PBS and incubated with secondary antibodies. Antibodies were diluted in PBS supplemented with 2.5% (w/v) milk and 0.3% (v/v) Tween 20. Membranes were washed with 0.3% (v/v) Tween 20 in PBS, with PBS, and subsequently scanned with Odyssey system (LI-COR Biotechnology). Protein band intensities were quantified using Fiji as the area under the curve of each band. All cellular and viral markers were normalised by dividing their intensity by the corresponding beta-actin intensity.