Anti ho 1 monoclonal antibody

1.6 × 10⁵ cells from each line were seeded into the wells of six-well plates (Costar, Corning, NY). After overnight culture, each well received 1.6 mL of the appropriate culture medium containing 12.5 μg/mL NPs for an additional 6 h. Untreated cells were used as a blank control, while TiO₂ NPs were used as a negative control. Cells were washed with PBS three times and harvested by scraping. The cell pellets were resuspended in cell lysis buffer containing Triton X-100 and protease inhibitors. After sonication and centrifugation, the protein content of the supernatants was measured by the Bradford method and 30 μg protein from each sample was electrophoresed by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking, the membranes were incubated with anti-HO-1 monoclonal antibody (1:1000) (ENZO Life Sciences, Plymouth Meeting, PA) or anti-metallothionein monoclonal antibody (1:1000) (Abcam, Cambridge, MA). The membranes were overlaid with biotinylated secondary antibody (1:1000) before the addition of the HRP-conjugated avidin-biotin complex (1:10 000). The proteins were detected using the ECL reagent according to the manufacturer's instructions.