Chemi xrs system

Confluent monolayers of HUVECs were treated with TNFα, IL-1β, or LPS, as indicated in Results and Figure Legends. At the end of the treatment period, cells were lysed in ice-cold cell lysis buffer (50 mmol/L HEPES, 2 mmol/L EDTA, 150 mmol/L NaCl, and 1% Triton X-100) containing protease and phosphatase inhibitors single-use cocktail (Thermo Fisher). Cell lysates were incubated with the Gab2 polyclonal antibodies (Sigma) overnight at 4 °C. After that, 20 µL of protein A/G agarose beads were added to the reaction mixture and incubated at room temperature for 2 to 3 hours with constant rotation. The immunocomplexes were sedimented by centrifugation at 150g for 8 minutes at 4 °C. The pelleted agarose beads were washed 3× with the cell lysis buffer to remove the unbound material. The bound material was eluted by adding SDS-PAGE sample buffer (25 µL) to the beads and heating the sample at 95 °C for 15 minutes. Where no immunoprecipitation was involved, the cells were lysed directly in the SDS-PAGE sample buffer. An equal amount of protein or volume was subjected to SDS-PAGE and processed for immunoblot analysis to probe specific signaling proteins. The immunoblots were developed with chemiluminescence using Western Lightning Plus HRP substrate (Millipore). Densitometric analysis was performed using the Bio-Rad Chemi XRS system and Image J software.