2.5. Immunoprecipitation

A two-step immunoprecipitation method was used as described earlier [23,24]. For pre-clearing, cell lysates were incubated with 5 µL of normal horse serum and 30 µL of protein A/G-conjugated Sepharose beads for 1 h at 4 °C. For antibody coupling, pre-cleared cell lysates were separated from beads by centrifugation at 7000× g for 5 min at 4 °C. Supernatants were further incubated with 2.5 µL of NF-κB p105/50 antibody (Cell Signaling, Beverly, MA, USA, cat ##3035) and 100 µL of the protein A/G gel beads for overnight at 4 °C. Samples were allowed to centrifuge at 12,000× g for 15 min at 4 °C to separate antigen–antibody complexes. After washing with lysis buffer, complexes were later eluted in 30 µL of SDS-sample buffer. Five microliter aliquots of each sample were used to run 8% SDS-PAGE resolving gel followed by western blots using TLR2 (Abcam, Cambridge, UK, cat #ab213676) or iNOS (Abcam, Cambridge, UK, cat #ab3523) primary antibody. Appropriate HRP-conjugated secondary antibodies were used for 1 h at RT and developed by using ECL substrate kit as described before [24].