Anti cd55

Protein samples were prepared from S. aureus-infected HaCaT cells using Laemmli buffer and run with 12% (w/v) SDS-PAGE in a Glycine/Tris/SDS buffer. The separated proteins were transferred onto a polyvinylidene difluoride membrane for 2 h at 100 V, preincubated in blocking buffer (5% (w/v) nonfat dried milk in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBS-T)) for 2 h at RT, and then incubated overnight at 4 °C with primary antibodies (diluted to 1:1000 in TBS-T): anti-CD55, anti-ULBP1, anticlaudin-1, antioccludin, anti-Zo-1 (These were purchased at Santa Cruz Biotechnology), anti-MAP kinase (R&D System), anti-BCL2 (Abcam, Cambridge, MA, USA), anticaspase 1 (Abcam), and anticaspase 7 (Cell Signaling Technology, MA, USA). The membrane was then washed 3 times with TBS-T and reacted for 2 h with HRP-conjugated secondary antibodies (diluted to 1:2000 in TBS-T; Santa Cruz). After three more washes with TBS-T, the protein bands were emitted using an ECL Pico Western blotting reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) and detected with X-ray film. β-actin was used as a loading control.