Odessy scanner

After incubating A549, H1975, and HCC827 cells with P.A for the indicated amount of time, cells were harvested and washed with cold 1× PBS. Then, cells were lysed with ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors to extract the cellular proteins. Total extracted protein was quantified using the Bio-Rad DCTM protein assay kit (Bio-Rad, Philadelphia, PA, USA). Then, 35 μg of each protein sample was loaded and electrophoretically separated on an 8% SDS-PAGE gel and then transferred to a Nitrocellulose (NC) membrane. Membranes were blocked with 5% non-fat milk and PBS containing 0.1% Tween-20 (TBST) for 1 h at RT. After 1 h, membranes were incubated with primary antibodies (1:1000 dilution) at 4 °C overnight with gentle shaking. Membranes were washed three times with TBST (5 min/time) and incubated with a secondary fluorescent antibody (1:10,000 dilution) for 1 h at RT. After another three washes with TBST (15 min/time), bands were visualized on a LI-COR Odessy scanner (Belfast, ME, USA).

To assess expression of viral proteins (p24 and p17) or to validate knockdown of host proteins (MAVS, STING, RIG-I, and MDA5), cell lysates containing 20−30 µg total protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: a mouse anti-p24 antibody (p24-2, Dr. Michael H. Malim, NIH AIDS Reagent Program, #6457, 1:5000) or a rabbit anti-p24 antibody (ImmunoDX, #1303, 1:1000), a rabbit anti-p17 polyclonal antibody (VU47, Dr. Paul Spearman and Dr. Lingmei Ding, NIH AIDS Reagent Program, #4811, 1:1000), a rabbit anti-MAVS polyclonal antibody (Thermo Fisher, #PA5-17256, 1:1000), a rabbit anti-STING polyclonal antibody (Cell Signaling, #13647, 1:1000), a mouse anti-RIG-I antibody (Enzo Life Sciences, #ALX-804-849-C100, 1:1500) or a rabbit anti-MDA5 antibody (Proteintech, #21775-1-AP, 1:1000). Then, the membranes were stained with secondary antibodies, a goat anti-mouse-IgG-DyLight 680 (Pierce, # 35518, 1:10,000) or a goat anti-rabbit-IgG-DyLight 800 (Pierce, # 35571, 1:10,000). As loading controls, actin was probed using a rabbit anti-actin antibody (SIGMA, # A2066, 1:5000) or a mouse anti-actin antibody (Thermo Fisher, #AM4302, 1:5000). Membranes were scanned with an Odessy scanner (Li-Cor). All uncropped images are shown in Supplementary Fig. 7.