Protein extraction and Western blot

Total protein was extracted via RIPA buffer (Santa Cruz, CA) for the measurement of tight junction-associated proteins, and nuclear protein was extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, Rockford, IL) to detect the alteration of HIF-1α expression after HaCaT cells were exposed to Nano-Ni or Nano-TiO₂. The protein concentration was determined by the Bradford method using a spectrophotometer (Beckman Coulter, Brea, CA). 30 μg total protein or 20 μg nuclear protein of each sample was separated on 10% or 7.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). After blocking with 5% skim milk/TBST (50 mM Tris-HCl, 140 mM NaCl, 0.05% Tween-20, pH 7.5) for 2 h at room temperature, the membranes were then incubated with primary antibodies at 4 °C overnight. After washing, the membranes were incubated with horse anti-mouse or goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 2 h. Immunoreactive bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) followed by exposure to CL-XPosure™ film (Thermo Scientific). Equal protein loading was verified by Coomassie Brilliant Blue staining. The expression of β-actin was used as an internal reference. Immunoreactive bands were quantified by NIH ImageJ software (http://imagej.nih.gov/ij/).