Nfkb p65 total phospho human instantone elisa kit

MDMs were infected or not with P. aeruginosa (MOI 30) in the presence or absence of INH172 and stimulated or not with ABL/PI5P at a ratio of 1:1 (ABL:MDM) for 2 h. Thereafter, supernatants were collected, cells were lysed, and both stored at −20°C until analysis. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and transforming growth factor-beta (TGF-β) in the supernatants of MDMs were measured by human TNF-α ELISA kit (BD Biosciences), human IL-6 DuoSet^® ELISA Development Systems, human IL-1β DuoSet^® ELISA Development Systems, human IL-10 DuoSet^® ELISA Development Systems, and human TGF-β DuoSet^® ELISA Development Systems (all by R&D system) and used according to the manufacturer’s instructions. The levels of murine TNF-α, KC, JE, and MIP-2 were measured by DuoSet^® ELISA Development Systems (R&D system). The activation of NF-κB transcription factor was assessed on lysed cells by “NFkB p65 (Total/Phospho) Human InstantOne™ ELISA Kit” (Invitrogen) and used according to the manufacturer’s instructions.

5 × 10⁵ cells/well of HaCaT cell line was seeded on the 96-well plate. After overnight incubation at 37 °C, the media was removed and 100 µL of each sample extract at a concentration of 25 mg/ml was added into the well separately and incubated for another 24 h. The amount of total NF-kB p65 and of phosporylated NF-kB p65 proteins presented in the cell lysate was then quantified using an Invitrogen™ NF-kB p65 (Total/Phospho) Human InstantOne ELISA kit, following the manufacturer’s instruction.

The 5 × 10⁵ cells/well of HaCaT cell line was seeded on the 96-well plate. After overnight incubation at 37 °C, the media was removed and 100 µL of each sample extract at a concentration of 25 mg/mL was added into the well separately and incubated for another 24 h. The amount of total NF-kB p65 and of phosporylated NF-kB p65 proteins presented in the cell lysate was then quantified using an Invitrogen NFkB p65 (Total/Phospho) Human InstantOne ELISA kit, following the manufacturer’s instructions.