Western Blotting and NF-κB p65 Transcription Factor Assay

HaCaT cells were treated with 10 μg/mL Der p 38 for the indicated time, and then harvested cells were lysed in lysis buffer (TransLab, Daejeon, Korea). The homogenate was centrifugated at 12,000 x g for 15 min at 4°C, and the supernatant was collected as total lysate. Protein concentration of the lysate was measured by a protein assay kit (Thermo Scientific). Following separation of the protein samples (50 μg/lane) by 10% SDS-PAGE, the transferred nitrocellulose membrane was sequentially incubated with primary (1:1,000) and secondary antibody (1:3,000) for 1 h at room temperature, and developed using the Enhanced Chemiluminescence Western blotting Detection System (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The same blot was stripped and re-probed with internal control antibodies, such as anti-ERK2 antibodies. For evaluating NF-κB activity, nuclear lysates were assessed for NF-kB DNA-binding activity using NF-κB p65 transcription factor assay kit (Abcam, Cambridge, UK) as described in our previous paper (24).