Freshly isolated hmoDCs were previously pre‐incubated with Pepinh‐MYD or Pepinh‐control for 6 h and with Gefitinib or DMSO for 30 min before the stimulation with control or MV130 for 30 min. Cell lysis was carried out with PBS/Triton 1% in the presence of 1 mM PMSF (Sigma), 1 μg/mL Leupeptin (Bachem) and Aprotinin (Roche) for 30 min at 4 °C with vortex every 10 min. Lysates were clarified by centrifugation at 10 000 × g for 15 min at 4°C. Protein quantification was performed with Micro BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions and samples with equal amounts of total protein were resolved in 10% SDS‐PAGE. Proteins were then transferred to nitrocellulose membranes.
The membranes were incubated with the following antibodies: anti‐human phospho‐IkBα (Ser32/36) (1/1000, Cell signalling), anti‐human phospho‐IKKα/IKKβ (Ser176/Ser177) (1/1000, Cell signalling), and anti‐human β‐actin (1/5000, Sigma) as primary antibodies and Goat anti‐mouse or anti‐rabbit conjugated with horseradish peroxidase (1/2500, Pierce) as secondary antibodies. Reactive bands were visualized by using ECL chemiluminescence system (BioRad). Optical density of the reactive bands was quantified with Fujifilm multigauge software and values expressed relative to β‐actin loading control.
The membranes were incubated with the following antibodies: anti‐human phospho‐IkBα (Ser32/36) (1/1000, Cell signalling), anti‐human phospho‐IKKα/IKKβ (Ser176/Ser177) (1/1000, Cell signalling), and anti‐human β‐actin (1/5000, Sigma) as primary antibodies and Goat anti‐mouse or anti‐rabbit conjugated with horseradish peroxidase (1/2500, Pierce) as secondary antibodies. Reactive bands were visualized by using ECL chemiluminescence system (BioRad). Optical density of the reactive bands was quantified with Fujifilm multigauge software and values expressed relative to β‐actin loading control.