Genesnap software

Western blotting was done using our established protocol (Iwasaki et al. 2016), and 40-μg protein samples were loaded to each well. Briefly, after electrophoresis and transfer onto a polyvinylidenedifluoride (PVDF) membrane using the iBlot® 2 Dry Blotting System (ThermoFisher scientific), membranes were blocked with 5% non-fat powdered milk in Tris-buffered saline with Tween (TBS-T) for 1 h at room temperature, and then incubated overnight at 4 °C with anti-TIMP-2 mouse primary antibody (Santa Cruz Biotechnology), anti-HIF1α (Novus biologicals), anti-E-cadherin (Santa Cruz Biotechnology) or anti-GAPDH mouse antibody (Millipore), followed by horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse (Cell Signaling technology) secondary antibody for 1 h. Protein bands were visualised using the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology) and the Syngene GeneSnap software (Syngene, Cambridge, UK). Western blotting band quantification was conducted with software ImageJ (https://imagej.nih.gov/ij/) relative to GAPDH expression.

Samples were resolved on 8% SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad), and incubated with primary antibodies against STEP (1:1000, Millipore), non-phospho-STEP (1:1000, Cell Signaling), phospho-Tyr (1:2000, Millipore), GluN2A (1:2000, Millipore), GluN2B (1:2000, Millipore), pY416 Src (1:1000, Cell Signaling), Src (1:2000 Santa Cruz), Fyn (1:2000 Santa Cruz), and β-actin (1:5000, Santa Cruz) overnight at 4 °C, and HRP-conjugated secondary antibodies (1:5000; Pierce) or Clean-Blot IP detection reagent HRP (1:1000; Thermo Scientific, Rockford, IL) for 2 h at RT. Immunoreactivity was developed with a Chemiluminescent Substrate kit (Pierce) and visualized by G:BOX with the GeneSnap software (Syngene, Cambridge, UK). All densitometric quantification was obtained using ImageJ software (National Institutes of Health).

For each sample, 50 µg of renal tissue protein extract was used for electrophoresis on 10% polyacrylamide gels (SDS-PAGE). The immunostaining was carried out with primary antibodies (β-actin/1:1,000, Sigma, USA; IKK-α/1:1,000, Cell Signaling, USA; Argonaute 2/1:1,000, Cell Signaling, USA; Drosha/1:1,000, Cell Signaling, USA; Dicer/1:1,000, Imgenex, India), followed by conjugated secondary antibodies (anti-mouse or anti-rabbit peroxidase/1:5,000, Sigma, USA). Then, the membrane was revealed by chemiluminescence methods using the ECL kit (Millipore, USA), and the images were acquired on GEN-BOX equipment (Syngene, UK) (n = 5). The GeneSnap software and GeneTools (Syngene, UK) were used to identify, analyze, and quantify the gel bands.

A549 and MDCK cells were seeded on 96-well plates with 2.5 × 10⁴ cells/well. One day later, cells were infected at a MOI of 3 with 50 μL of inoculum. Following 1 h of adsorption at 35°C, cells were washed twice with DMEM and incubated with 100 μL of DMEM containing 10% FCS. Plates were then incubated at 35°C for 3, 6, or 9 h. Protein extracts were prepared in Laemmli buffer, separated on 4–12% NuPAGE Bis-Tris gel (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes (Hybond^®, Amersham). Immunoblot membranes were incubated with primary antibodies directed against NP (AAH5, Abcam, 1/5,000 for IAV; B017, Abcam, 1/1,000 for IBV) or GAPDH (Pierce) and revealed with peroxidase-conjugated secondary antibodies (GE Healthcare) and the ECL2 substrate (Thermo Fisher Scientific). The chemiluminescence signals were acquired using a G-Box and the GeneSnap software (SynGene).

Following total protein extraction, western blotting was performed as previously described [30 (link)]. Primary antibodies used in this study were as follows: rabbit anti-Akt (Cat. # 9272), pAkt (S473, Cat. # 9271), LKB1 (Cat. # D60C5) and PARP (Cat. # 9542) antibodies were from Cell Signaling Technology (Danvers, MA), mouse anti-FAK (clone 77/FAK) was from BD Transduction Laboratories (Mississauga, ON), and mouse anti-β-actin antibody (clone AC-74) was from Sigma (St. Louis, MO). Following incubation with primary antibody overnight, membranes were washed 3 x 5 minutes and incubated in horse radish peroxidase (HRP) conjugated goat anti-mouse or goat anti-rabbit secondary antibody (Calbiochem, San Diego, CA) for 1 hour. Membranes were then washed 6 x 5 minutes and incubated in Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA) prior to image development using the GeneGnome Bio Imaging System and GeneSnap software (Syngene, Frederick, MD).