The Staphylococcus aureus iron-regulated surface determinant A (IsdA) increases SARS CoV-2 replication by modulating JAK-STAT signaling WESTERN BLOTS
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western blot images related to Figure S3, S6 and S8.
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For detection of secreted bacterial proteins, bacteria were grown overnight in TSB, bacteria were pelleted and the supernatant (equal to OD600 of 8) was used for a trichloroacetic acid (TCA) precipitation. Briefly, equal volumes of bacterial supernatant and 20% (w/v) TCA were mixed and incubated at 4˚C for 3h. Samples were pelleted at 21 000 x g for 15 min, washed twice with ice-cold 70% ethanol and allowed to dry overnight. Pellets were re-suspended in 40 µL Laemmli buffer, boiled at 95˚C for 10 min and 15 µL loaded on a 12% SDS-PAGE gel. For detection of host and viral proteins, 6 wells of a 12 well tissue culture plate of Vero E6 cells were lysed in a total volume of 450 µl of RIPA buffer (75µl per well) containing a Complete Mini protease inhibit tablet (Roche). Samples were incubated on a rocker for 30 min, followed by centrifugation at 12 000 x g for 20 minutes. The resulting supernatant was harvested and mixed with equal volume of 2x Laemmli buffer, boiled at 95˚C for 10 min and 15 µL loaded on a 10% SDS-PAGE gel. Samples were run at 150V for 90 min and transferred on a nitrocellulose membrane using a TransBlotter Turbo (Biorad) standard settings. Membranes were blocked in 7% (w/v) skimmed milk in PBS + 0.1% Tween-20 (PBST) overnight at 4˚C. Primary antibody was added in blocking buffer overnight at 4˚C, followed by 3 washes with PBST. Secondary antibody was added at 1 in 20 000 dilution in PBST for 1h, followed by 3 washes with PBST. Membranes were imaged on a LiCor scanner. Antibodies used were: anti S. aureus IsdA polyclonal serum (1:500 dilution), Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (NEB 9145S) (1:500 dilution), STAT3 Rabbit mAb (Abclonal A19566) (1:500 dilution), β-Actin Mouse mAb (Abclonal AC004) (1:1000 dilution), SARS-CoV-2 N Protein Rabbit mAb (Abclonal A20021) (1:1000 dilution). Secondary antibodies were IRDye 800CW donkey anti rabbit and IRDye 680RD goat anti-mouse (Licor).