Protein a g plus agarose

For CO-IP, cells were lysed in Pierce IP lysis buffer (No. 87787, Thermo Fisher) and protease inhibitor cocktail (No. 04693159001, Roche). Cell lysates (500 μl) were incubated with anti-HMGB1 antibody (ab18256, 1:200, Abcam) or control IgG (40 μl Protein A/G PLUS-Agarose, No. 17061801, GE health) overnight at 4 °C. The beads were washed three times with PBS, followed by immunoblotting. Immunoblotting was performed as previously described [31 (link)]. Primary antibodies used were anti-HMGB1 (ab18256, 1:1000, Abcam), anti-SQSTM1/p62 (88588S, 1:1000, CST), anti-ATG5 (12994 T, 1:1000, CST), anti-ATG7 (8558 T, 1:1000, CST), anti-LC3B (sc-376404, 1:1000, Santa), anti-GAPDH (5174S, 1:1000, CST), anti-RAGE (ab3611, 1:1000, Abcam), anti-GORASP2 (10598–1-AP, 1:1000, Proteintech), anti-p-ERK1/2 (8544S, 1:1000, CST), anti-IKB (9246S, 1:1000, CST), anti-NLRP3 (19771–1-AP, 1:1000, Proteintech), anti-ASC (sc-514414, 1:1000, Santa) and anti-p-NF-κB p65 (3033S, 1:1000, CST), anti-β-TUBULIN (2128S,1:1000, CST).

To verify the PPIs identified by AP-MS, mCherry-Strep-tagged effectors and FLAG-tagged host proteins were co-expressed in 293T cells. For identification of the interaction between CirB and PSMB5, different tagged CirB and PSMB5 proteins, as well as their truncation mutants, were co-expressed in HeLa cells. Twenty-four hours post transfection, cell lysates were prepared with lysis buffer, and the supernatant of lysates after centrifugation was incubated with 2 μg of anti-FLAG antibody (Sigma, F1804) at 4°C for 6 hours. Then, 50 μl of a 50% slurry consisting of Protein A/G plus-agarose (GE Healthcare, 17061801) was added, mixed and incubated for another 6 hours or overnight. After 5 washes with lysis buffer, the beads were boiled with 2×loading buffer for 10 min. The samples were fractionated by electrophoresis on SDS-PAGE gels, and the resolved proteins were transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were incubated with the indicated antibodies, followed by appropriate secondary antibodies. Blots were developed with an enhanced chemiluminescence (ECL) kit.