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Fig. 1 | BMC Biology

Fig. 1

From: Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

Fig. 1

Paxillin binds directly to the LRR domain of NLRP3 in the cytosol. a Identification of NLRP3 LRR domain-Paxillin interaction by yeast two-hybrid analysis. Yeast strain AH109 cells were transformed with the combination of BD and AD plasmid, as indicated (1–3). Transformed yeast cells were first grown on the SD-minus Trp/Leu plates for 3 days. The colony of yeast was then streaked on SD-minus Trp/Leu/Ade/His plates (QDO). BD-p53 and AD-T (5) was used as a positive control and BD-Lam and AD-T (4) as a negative control. b HEK293T cells were co-transfected with pHA-Paxillin and pFlag-Vector, pFlag-NLRP3, pFlag-ASC, and pFlag-Casp-1. Lysates were prepared and subjected to IP using an anti-Flag antibody and analyzed by immunoblotting using an anti-HA or anti-Flag antibody (top) or subjected directly to Western blot using an anti-Flag or anti-HA antibody (as input) (bottom). c HEK293T cells were co-transfected with pHA-Paxillin and pFlag-Vector, pFlag-NLRP3, pFlag-PYRIN, pFlag-NACHT, and pFlag-LRR. Lysates were prepared and subjected to IP using an anti-Flag antibody and analyzed by immunoblotting using an anti-HA or anti-Flag antibody (top) or subjected directly to Western blot using an anti-Flag or anti-HA antibody (as input) (bottom). d Extracts from HEK293T cells transfected with Flag-Paxillin were incubated with 10 μg GST proteins or GST-LRR protein that was incubated with glutathione-Sepharose beads. The mixture was washed three times and then analyzed by immunoblotting using anti-Flag and anti-GST antibody (top). Lysates from transfected HEK293T cells were analyzed by immunoblotting using anti-Flag antibody (as input) (bottom). e Extracts from HEK293T cells transfected with Flag-NLRP3 were incubated with 10 μg GST proteins or GST-Paxillin protein which was incubated with glutathione-Sepharose beads. The mixture was washed three times and then analyzed by immunoblotting using anti-Flag and anti-GST antibody (top). Lysates from transfected HEK293T cells were analyzed by immunoblotting using anti-Flag antibody (as input) (bottom). f HeLa cells were transfected with pFlag-Paxillin and pHA-NLRP3 or co-transfected with pFlag-Paxillin and pHA-NLRP3. Subcellular localization of Flag-Paxillin (green), HA-NLRP3 (red), and the nucleus marker DAPI (blue) was examined by confocal microscopy

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