Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation
Fig 5
NLRP3 directly interacts with NleA.
(A) NleA associates with endogenous NLRP3. Columns packed with resins of bacterial expressed MBP (8 μg) or MBP-NleA (8 μg) were used to pulldown endogenous proteins of the THP-1 lysates prepared from approximately 1x108 cells. The bound proteins were eluted with a high salt elution buffer and analyzed with the indicated antibodies. (B) NleA interacts with NLRP3 in cells. HeLa cells were seeded in 6-well plates and transfected with plasmids expressing the indicated fusion proteins for 24 hrs. The cell lysates were immunoprecipitated (IP) with anti-GFP-coupled magnetic beads. The pulldown products were analyzed by immunoblotting (IB) using anti-NLRP3 and anti-GFP antibodies. (C) NleA directly binds NLRP3. Purified GST or GST-NLRP3-bound resins (2.5 μg each) were packed in columns to pull down the purified MBP-NleA (250 μl of 5 μg/ml). The bound proteins were eluted with a high salt elution buffer and analyzed by IB using an anti-MBP antibody. (D) Domains of NLRP3. The amino acid sequences containing one of three domains were fused with GST for purification. (E) NleA binds to the PYD and LRR domains of NLRP3. GST or GST-PYD, GST-NACHT, and GST-LRR fusion proteins were purified and immobilized on resins at approximately 2.5 μg each. An equal amount of purified MBP-NleA (500 μl of 5 μg/ml) was applied for direct binding. The columns were extensively washed with column buffer. The eluted products were probed with an anti-MBP antibody.