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. 2016 Nov 28;36(24):3075–3085. doi: 10.1128/MCB.00450-16

FIG 3.

FIG 3

The association between HBZ and CUL1 occurs by a head-to-tail interaction. (A) HEK293T cells were cotransfected with 3 μg of a plasmid expressing FLAG-CUL1 with (+) or without (−) 3 μg of a plasmid expressing the HBZ unspliced form-HA (-US) or the HBZ spliced isoform-HA (-SI). After 36 h, cell extracts were prepared and subjected to immunoprecipitation using an anti-HA antibody, followed by immunoblot analysis with anti-FLAG and anti-HA antibodies. Total protein levels in whole-cell lysates were analyzed by immunoblotting using anti-FLAG, anti-HA, and anti-α-tubulin antibodies. (B) Schematic diagram of full-length CUL1 (CUL1-full) and the CUL1 deletion mutants (CUL1-A, CUL1-B, and CUL1-C) used in this study. Characteristic domains of CUL1 are indicated. (C) Schematic diagram of full-length HBZ (HBZ-full) and the deletion mutants (HBZ-N1, HBZ-N2, and HBZ-C) used in this study. Characteristic domains of HBZ are indicated. (D) HEK293T cells were cotransfected with 3 μg of a plasmid expressing HBZ-HA with or without 3 μg of a plasmid expressing Myc-full-length CUL1, CUL1-A, CUL1-B, or CUL1-C. After 36 h, cell extracts were prepared and subjected to immunoprecipitation using an anti-HA antibody, followed by immunoblot analysis with anti-Myc and anti-HA antibodies. Total protein levels in whole-cell lysates were analyzed by immunoblotting using anti-Myc, anti-HA, and anti-α-tubulin antibodies. (E) HEK293T cells were cotransfected with 3 μg of a plasmid expressing Myc-CUL1 with or without 3 μg of a plasmid expressing full-length HBZ-HA, HBZ-N1-HA, HBZ-N2-HA, or HBZ-C-HA. After 36 h, cell extracts were prepared and subjected to immunoprecipitation using the anti-HA antibody, followed by immunoblot analysis with anti-Myc and anti-HA antibodies. Total protein levels in whole-cell lysates were analyzed by immunoblotting using anti-Myc, anti-HA, and anti-α-tubulin antibodies. (F) HEK293T cells were cotransfected with 3 μg of a plasmid expressing Myc-CUL1-A with or without 3 μg of a plasmid expressing HBZ-full-FLAG, HBZ-N1-FLAG, or HBZ-C-FLAG. After 36 h, cell extracts were prepared and subjected to immunoprecipitation using an anti-Myc antibody, followed by immunoblot analysis with anti-FLAG and anti-Myc antibodies. Total protein levels in whole-cell lysates were analyzed by immunoblotting using anti-FLAG, anti-Myc, and anti-α-tubulin antibodies. (G) HEK293T cells were cotransfected with 3 μg of a plasmid expressing Myc-CUL1-C with or without 3 μg of a plasmid expressing HBZ-full-HA, HBZ-N1-HA, or HBZ-C-HA. After 36 h, cell extracts were prepared and subjected to immunoprecipitation using the anti-HA antibody, followed by immunoblot analysis with anti-Myc and anti-HA antibodies. Total protein levels in whole-cell lysates were analyzed by immunoblotting using anti-Myc, anti-HA, and anti-α-tubulin antibodies. *, nonspecific bands; WCL, whole-cell lysates; IB, immunoblotting; IP, immunoprecipitation.