Trpm8

24 h after menthol aerosol exposure in the VITROCELL® cloud chamber, cells in transwells were lysed using RIPA buffer. To evaluate NF-κB activation, nuclear and cytoplasmic fractions of cell suspensions were isolated after treatment using the Nuclear/Cytosol Fractionation Kit (Thermo Fischer Scientific). Western blotting was performed as previously described (Nair et al., 2014 (link)). Lysates were vortexed every 15 min for 1 h, centrifuged at 10,000 × g for 10 min at 4°C, quantified using the Pierce BCA assay kit (Thermo Scientific, Waltham, Massachusetts). Lysates were mixed with Laemmli buffer (1:4), 20 μg of protein/gel lane were separated using SDS gel electrophoresis (100 V for 2 h), and then transferred to a PVDF membrane (BioRad, Carlsbad, CA) by wet electroblotting. The membrane was blocked (5% milk in TBST buffer x 45 min) and incubated overnight at 4°C with antibodies against TRPM8 (Abcam, Cambridge, MA), SOD2 (Cell Signaling Technology, Danvers, MA), or β-actin (Cell Signaling Technology, Danvers, MA). Membranes were washed for 30 min in TBST, incubated in secondary antibody for 2 h, then developed using immunoblot reagent (Bio ad, Hercules, California ) in a ChemiDoc™ Imaging ystems (Bio ad, Hercules, California ).