Las 3000 developer

For protein detection, cells grown for 3 days in a 25 cm² flask were washed with PBS and extracted with 150 µl 1% SDS in TE prior to protein quantification by bicinchoninic acid (BCA) (Pierce). Fifty micrograms of total protein were loaded per gel, and a western blot was performed using M2 anti-Flag mouse monoclonal antibody (Sigma, Cat No. F3165, 1:2000 dilution) or anti-GFP monoclonal antibody (Roche, Cat No. 11814460001, 1:2000 dilution) and detected with anti-mouse IgG conjugated to horseradish peroxidase (Jackson, Cat No. 515035003, 1:10,000 dilution). Blots were developed using high-sensitivity electrochemiluminescence (ECL) reagent (Thermo Fisher Scientific) and visualized using the Fujifilm LAS-3000 developer. Intensities of immunoreactive bands on western blots were quantified using Quantity One (Bio-Rad) software. Protein levels were normalized using a ribosomal protein (RL7) antibody as a loading control^{30 (link)} (1:500 dilution).

HmoDCs (10⁶ cells per ml) were treated with control excipients, V132 (3 FTU per ml), MV140 (10⁷ bact. per ml) or MV140/V132 (MV140, 10⁷ bact. per ml, and V132, 3 FTU per ml). After 30 min at 37°C, cells were harvested and lysed with RIPA buffer (ThermoFisher scientific) in presence of Protease/Phosphatase Inhibitor Cocktail (Cell Signaling) for 30 min at 4°C vortexing every 10 min. Lysates were clarified by centrifugation at 10,000g for 15 min at 4°C. Protein quantification was performed with Micro BCA Protein Assay Kit (Pierce) and samples with equal amounts of total protein were resolved in 10% SDS-polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were then transferred to nitrocellulose membrane (BioRad). The membrane was incubated with the primary antibodies: phospho-ERK1/2 (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-IKKα (Ser176)/IKKβ (Ser177), or phospho-Akt (Ser473) (1:1000, Cell Signaling), phospho-p70 S6 Kinase (Thr389) (1:750; Cell Signaling), and β-actin (1:15000, Sigma-Aldrich); and goat anti-rabbit (1:4000, BioRad) or goat anti-mouse (1:2500, Pierce) conjugate with horseradish peroxidase as a secondary antibody. The signal was developed with Clarity Western ECL Substrate (Bio-Rad) and detected in a Fujifilm LAS-3000 developer.