Mouse monoclonal anti beta actin antibody

Approximately 12 ug of eye and brain lysate and 20 ug of joint lysate were loaded per well of 4–12% Bis-Tris gels (ThermoFisher scientific). After separation, proteins were transferred onto nitrocellulose membranes using the iblot system (ThermoFisher scientific). Membranes were blocked using TBS with 5% milk prior to overnight incubation with 5 ng of mouse monoclonal anti-beta actin antibody, 11 ng rabbit anti mouse HSP70 and 10 ng rabbit anti mouse creatine kinase, all from Abcam. After extensive washes, membranes were incubated with 1 ng of Dylight 800 conjugated goat anti-mouse IgG and 1 ng Dylight 680 conjugated goat anti-rabbit IgG (Mandel scientific) and analyzed as described above.

T-47D cells grown at 1×10³ cells per well in 96-well plates (Corning Incorporated Costar, #3603). After 24 h incubation, chemical compounds (10 μM) were added to each well. After 48 h incubation, plates were fixed with 4% paraformaldehyde (PFA) for 20min at room temperature (RT). Then the cells were permeabilized with 0.2% Triton X-100 for 15min at RT, and blocked with Odyssey Blocking Buffer (TBS) (LI-COR, 9275000) for 1 h at RT. The cells were incubated at 4°C overnight with a rabbit monoclonal anti-ORF1p antibody (diluted 1:200, Abcam) and a mouse monoclonal anti-beta-actin antibody (diluted 1:250, Abcam) with gentle shakes. After five washes with PBS, the cells were stained with an IRDye^® 800RD Goat anti-Rabbit antibody (diluted 1:400, LI-COR, #926–68071) and IRDye^® 680CW Goat anti-Mouse antibody (1:600, LI-COR, #926–32210) at RT for 2 h. The microplates, were scanned with the Odyssey system (LI-COR), and the integrated fluorescence intensities representing the protein expression levels were acquired using Image Studio Ver 5.2 software (Egorina et al., 2006 (link)). Compared with DMSO control, compounds with an inhibition rate over 50% were defined as promising compounds.

Viral NP protein was detected via western blot analysis. Briefly, total cellular proteins from H1N1pdm09-infected MDCK-II cells (MOI = 3), either binase-treated (1 × 10⁵ U/ml, 4 h before infection and during 8 h p.i.) or left untreated, were extracted at 4, 6, 8 h p.i. as described before [30 (link)]. The protein concentration of each sample was measured using Bradford Assay Kit (Bio-Rad, USA). Cell lysates were subjected to 4–12% SDS-polyacrylamide gradient gel electrophoresis (Invitrogen, USA) and subsequent Western blotting. The samples were transferred onto polyvinylidenefluoride (PVDF) membranes (GE Healthcare, UK) according to the manufacturer’s instructions using XCell II Blot Module (Invitrogen, USA). The NP protein of H1N1pdm09 was detected using a rabbit polyclonal anti-influenza A (H1N1) virus NP protein antibody (1:1000, Thermo Scientific, USA). Beta Actin was detected as loading control using mouse monoclonal anti-beta Actin antibody (1:5000, Abcam, UK). As secondary antibodies for detection goat anti-mouse IRDye 800 CW and goat anti-rabbit IRDye 680 CW (1:15,000, Abcam, UK) were used and detected with Odyssey imaging systems (LI-COR, USA). Protein quantification was performed using “Quantity one” software (Bio-Rad, USA). To detect the internalized binase for binase-treated and nontreated MDCK-II cells, we performed western blot analysis as described previously.