Protein a g ultralink resin beads

HEK293 cells or mESCs were co-transfected with epitope-tagged expression plasmids. 48 hr after transfection, cells were harvested with lysis buffer (20 mM Tris-HCl PH 7.9, 25% glycerol, 150mM NaCl, 1.5mM MgCl₂, 0.1% NP-40, 0.2 mM EDTA, 0.5 mM DTT) supplemented with protease inhibitor cocktail (1861279, Thermo Scientific) and phosphatase inhibitor cocktail (78427, Thermo Scientific) and then sonicated for 5 cycles with 5 sec on and 30 sec off at 4°C. After centrifugation at 13,000g for 2 min, the supernatants were used for immunoprecipitation (IP). The cell extracts were incubated with anti-Flag, -Myc or -HA antibody for 2 hr at 4°C, followed by incubation with 25 μl of Protein A/G Ultralink Resin beads (53133, Thermo Scientific) for 1 hr. The beads were washed four times with lysis buffer at 4°C. The cell extracts (input) and IP samples were then subjected to Western blot with anti-Flag, -Myc and/or -HA antibodies for detecting protein-protein interactions. For antibody information, see Table S2.

One day before the experiment, HPASMC and HPAEC were plated at 70–80% of confluence into 12-well plates and grown overnight in the growth media described above. Apoptosis of HPASMC was induced by culturing the cells in DMEM, containing a reduced level of glucose (0.1g/L). In HPAEC, apoptosis was induced by culturing cells in serum-free ECM. Apoptosis in both cell types was measured 48 hours after induction. For necrosis induction, both cell types underwent 3–4 freeze/thawing cycles, as published^{6 (link)}. Conditioned media collected from apoptotic or necrotic HPAEC or HPASMC was used to treat the corresponding naïve HPAEC or HPASMC for 0–6 hours. To confirm the relevance of the visualized monomeric and dimeric bands to HMGB1 as well as to validate the contribution of HMGB1 in the stimulation of TLR4 and RAGE, the necrotic conditioned media was mixed with 25u/ml of neutralizing anti-HMGB1 antibodies^{20 (link)} (clone 3E8, Biolegend, San Diego, CA, cat# 651402) and incubated for overnight. The next day, the protein A/G ultra link resin beads (Thermo Fisher, cat#53132) were added to bind HMGB1-IgG complex. The samples were rotated at 4°C for 2 hours and centrifuged (30sec at 1,000 g). The supernatant (conditioned media with depleted HMGB1) or original necrotic media were used to treat naïve HPASMC for 6 hours.