Suppression of TREX1 Deficiency-Induced Cellular Senescence and Interferonopathies by Inhibition of DNA Damage Response
Description
Uncropped images from western blots
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Immunoblot analysis Cells were collected and resuspended in lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5%NP40) supplemented with protease and phosphatase inhibitors (ThermoFisher). Cell lysates were then centrifuged at 14,000 rpm for 10 min at 4°C and the supernatants were collected for immunoblotting. The total protein concentration was determined by the BCA protein assay kit (Beyotime). Equal amounts of protein samples were subjected to SDS-PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany) using a semi-dry transfer system (Bio-Rad, CA, USA). The PVDF membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with the indicated antibodies. Membranes were probed with primary antibodies as follows: cGAS (1:1000 dilution), STING(1:1000 dilution), TBK1(1:1000 dilution), p-TBK1(1:1000 dilution), IRF3(1:1000 dilution), p-IRF3(1:1000 dilution), TREX1(1:5000 dilution), RB (D20) (1:1000 dilution), p-RB (1:1000 dilution), CHK2 (1:1000 dilution), and p-CHK2 (phospho T68) (1:1000 dilution), p53(1:1000 dilution), p-p53(1:1000 dilution), p21(1:1000 dilution), p16(1:1000 dilution), H2AX(1:1000 dilution), γ-H2AX(1:1000 dilution), GAPDH(1:5000 dilution), Tubulin(1:5000 dilution). The membranes were then washed with TBST buffer three times and incubated with Goat anti-Rabbit IgG H&L (IRDye® 800 CW, ThermoFisher) preabsorbed ab216773 and anti-mouse IgG H&L (IRDye® 680 CW, ThermoFisher) preabsorbed ab216776 secondary antibodies at 1:10,000 dilution for 1 h at room temperature. The protein signals were analyzed by luminescence.
Institutions
- Fujian Normal University