Hrp conjugated secondary goat anti rabbit

To identify proteins interacting with PLY, pull-down was performed on DC and THP-1 native cell lysates using recombinant PLY as the bait. Cells were lysed with native lysis buffer (Abcam) containing 1x protease inhibitors (Roche) on ice for 15 min. Briefly, lysate corresponding to 0.8 mg protein was precleared by incubating with Protein G-agarose beads (Pierce) for 30 min at 4°C. Subsequently, the precleared lysate was incubated with 1 μg PLY (Cusabio) for 1 hr at 4°C and then incubated with Protein G beads conjugated to mouse anti-PLY (Abcam) with gentle rotation overnight at 4°C. As a control, lysates were incubated with isotype antibody or beads alone to distinguish non-specific interactions. The beads were washed thrice with PBS and the bound proteins were eluted by boiling in NuPAGE LDS sample buffer for 5 min at 95°C. The eluted proteins were identified using mass spectrometry at the Science for Life Laboratory in Uppsala, Sweden. The protein identifications were based on at least two matching peptides of 95% confidence per protein. To confirm the interaction between PLY and MRC-1, western blotting was performed on the eluate. MRC-1 was detected using rabbit anti-human MRC-1 (Abcam) and HRP-conjugated secondary goat anti-rabbit (GE Healthcare).