Figure 4
Figure 4. Domains of sHBZ responsible for suppression of NF-κB p65. (A) Schematic diagram of HBZ and its mutants used in this study. Characteristic domains of HBZ are indicated as follows: activation domain (AD), central domain (CD) and basic leucine zipper domain (bZIP). (B) Analysis of HBZ deletion mutants for the effect on p65-mediated NF-κB activation. Jurkat cells were cotransfected with κB-Luc and phRL-TK, with or without 1 μg of pCMV-Tag 2-p65, and with 1 or 5 μg pME18Sneo-sHBZ or sHBZ mutant. After 48 hours, luciferase levels were measured. *P < .05; **P < .01. Error bars represent SD. (C) Determination of the region of HBZ responsible for the interaction with p65. 293FT cells were transfected with the indicated mycHis-sHBZ mutants along with the FLAG-p65 vector. Cell lysates were subjected to immunoprecipitation (IP) using anti–c-Myc followed by immunoblotting (IB) using anti-FLAG. The expression levels of p65 and sHBZ mutants were detected. (D) Mapping the region of the p65 protein necessary for the interaction with sHBZ. The schema of p65 deletion mutants has been shown. The locations of the Rel homology domain, the nuclear localization signal (NLS), and the transactivation domain are indicated. 293FT cells were transfected with mycHis-sHBZ along with full-length or mutant FLAG-p65. At 48 hours after transfection, total cell lysates were subjected to IP using anti-FLAG followed by IB using anti-His. (E) HBZ did not influence p65/p50 interaction. 293FT cells were transfected with the indicated expression vectors. Cell lysates were subjected to IP using anti–c-Myc or anti-FLAG followed by IB using anti-HA. The expression levels of p65, p50, and sHBZ were detected. (F) sHBZ colocalizated with p65 in the cell nucleus. HeLa cells were transfected with mycHis-sHBZ together with (panels iii-viii) or without (panels i,ii) FLAG-p65. sHBZ was detected using anti-MYC Cy3 antibody (panels i,iii,vi). p65 was detected using anti–Flag-biotin and secondary Streptavidin-Alexa 488 antibody (panels iv,vii). The overlay of sHBZ and p65 is shown (panels v,viii). DAPI (4,6 diamidino-2-phenylindole) was used to counterstain the nucleus (panel ii). (G) sHBZ decreased p65 DNA binding capability. 293FT cells were transfected with FLAG-p65 together with either mycHis-sHBZ or one of its mutants. Cell lysates were subjected to the enzyme-linked immunosorbent assay (ELISA)–based NoShift assay to measure the DNA binding capability of p65. The absorbance at 450 nm indicated the binding ability of p65 (top panel). The bottom panel shows the amount of p65 and sHBZ in the 20% of input for analysis. *P < .05; **P < .01. Error bars represent SD.

Domains of sHBZ responsible for suppression of NF-κB p65. (A) Schematic diagram of HBZ and its mutants used in this study. Characteristic domains of HBZ are indicated as follows: activation domain (AD), central domain (CD) and basic leucine zipper domain (bZIP). (B) Analysis of HBZ deletion mutants for the effect on p65-mediated NF-κB activation. Jurkat cells were cotransfected with κB-Luc and phRL-TK, with or without 1 μg of pCMV-Tag 2-p65, and with 1 or 5 μg pME18Sneo-sHBZ or sHBZ mutant. After 48 hours, luciferase levels were measured. *P < .05; **P < .01. Error bars represent SD. (C) Determination of the region of HBZ responsible for the interaction with p65. 293FT cells were transfected with the indicated mycHis-sHBZ mutants along with the FLAG-p65 vector. Cell lysates were subjected to immunoprecipitation (IP) using anti–c-Myc followed by immunoblotting (IB) using anti-FLAG. The expression levels of p65 and sHBZ mutants were detected. (D) Mapping the region of the p65 protein necessary for the interaction with sHBZ. The schema of p65 deletion mutants has been shown. The locations of the Rel homology domain, the nuclear localization signal (NLS), and the transactivation domain are indicated. 293FT cells were transfected with mycHis-sHBZ along with full-length or mutant FLAG-p65. At 48 hours after transfection, total cell lysates were subjected to IP using anti-FLAG followed by IB using anti-His. (E) HBZ did not influence p65/p50 interaction. 293FT cells were transfected with the indicated expression vectors. Cell lysates were subjected to IP using anti–c-Myc or anti-FLAG followed by IB using anti-HA. The expression levels of p65, p50, and sHBZ were detected. (F) sHBZ colocalizated with p65 in the cell nucleus. HeLa cells were transfected with mycHis-sHBZ together with (panels iii-viii) or without (panels i,ii) FLAG-p65. sHBZ was detected using anti-MYC Cy3 antibody (panels i,iii,vi). p65 was detected using anti–Flag-biotin and secondary Streptavidin-Alexa 488 antibody (panels iv,vii). The overlay of sHBZ and p65 is shown (panels v,viii). DAPI (4,6 diamidino-2-phenylindole) was used to counterstain the nucleus (panel ii). (G) sHBZ decreased p65 DNA binding capability. 293FT cells were transfected with FLAG-p65 together with either mycHis-sHBZ or one of its mutants. Cell lysates were subjected to the enzyme-linked immunosorbent assay (ELISA)–based NoShift assay to measure the DNA binding capability of p65. The absorbance at 450 nm indicated the binding ability of p65 (top panel). The bottom panel shows the amount of p65 and sHBZ in the 20% of input for analysis. *P < .05; **P < .01. Error bars represent SD.

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