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. 2022 Nov 3;96(22):e00954-22. doi: 10.1128/jvi.00954-22

FIG 8.

FIG 8

The H240R protein (pH240R) interacts with NLRP3 and inhibits its oligomerization. (A) pH240R interacts with NLRP3. HEK293T cells were transfected with pFlag-H240R. GST pulldown and immunoblotting analyses were performed with an anti-GST or anti-Flag MAb. (B) Colocalization of pH240R with NLRP3. HEK293T cells were cotransfected with pFlag-NLRP3 (1.0 μg) alone or together with pMyc-H240R (1.0 μg) and analyzed by laser confocal microscopy for NLRP3 and pH240R. Bars, 10 μm. (C) The mRNA levels of NLRP3 were not affected by overexpression of pH240R. HEK293T cells were cotransfected with pFlag-NLRP3 (1.0 μg) and increasing doses of pFlag-H240R (0, 0.5, 1.0, and 2.0 μg) into 12-well plates. The mRNA levels of NLRP3 were examined by RT-qPCR. (D) The expression of NLRP3 was not inhibited by overexpressed pH240R. HEK293T cells were cotransfected with pFlag-NLRP3 (1.0 μg) and increasing doses of pFlag-H240R (0, 0.5, 1.0, and 2.0 μg) into 12-well plates. The protein expression was examined by Western blotting. (E and F) Expression levels of NLRP3 are not affected by pH240R in ASFV-infected PAMs. PAMs were infected with ASFV-WT or ASFV-ΔH240R for 24 h. The mRNA levels and the protein expression of NLRP3 were determined by RT-qPCR and Western blotting. (G) The oligomerization of NLRP3 is inhibited by pH240R. HEK293T cells were cotransfected with the NLRP3- and ASC-expressing plasmid in combination with the empty or H240R-expressing plasmid for 36 h, the cells lysates were prepared in NP-40, and the pellets were washed with PBS three times and cross-linked using 4 mM disuccinimidyl suberate (catalog no. 21555; Invitrogen) for 30 min. (H) Schematic representations of the NLRP3 domains. (I) The PRR region of NLRP3 interacts with H240R. HEK293T cells were cotransfected with pFlag-H240R and the plasmids expressing the Myc-tagged full-length or truncated NLRP3 constructs. The cell lysates were subjected to co-IP analysis using an anti-Myc MAb. *, protein bands that could be the cleaved products of full-length NLRP3. (J) The oligomerization of NLRP3-D1 inhibited by pH240R. HEK293T cells were cotransfected with the NLRP3-, NLRP3-D1-, NLRP3-D2-, NLRP3-D3-, or NLRP3-D4-expressing plasmid in combination with the empty or H240R-expressing plasmid, and the oligomerization of the wild-type and the truncated NLRP3 in the transfected cells was analyzed by laser confocal microscopy. Bars, 10 μm. ns, not significant.