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. 2014 Jun 9;9(6):e99503. doi: 10.1371/journal.pone.0099503

Figure 7. PIAS1 terminal-truncated mutants lack necdin-dependent ubiquitination.

Figure 7

(A) Diagrams of PIAS1 terminal-truncated mutants. Abbreviations: FL, full-length; ΔNT, N-terminal deletion mutant; ΔCT, C-terminal deletion mutant. PIAS domain names are as in Fig. 2A. (B) Interactions of necdin with PIAS1 mutants. HEK293A cells were transfected with expression vectors for necdin (1 µg), Myc-tagged PIAS1 FL (6 µg), ΔNT (4 µg) and ΔCT (8 µg). Cell lysates were immunoprecipitated (IP) with anti-necdin antibody and immunoblotted (IB) with anti-Myc antibody. (C) Protein levels of PIAS1 terminal-truncated mutants. HEK293A cells were transfected with expression vectors for Myc-tagged PIAS1 mutants (FL, ΔNT, ΔCT; 3 µg each) and necdin (1 µg). (D) Ubiquitination of PIAS1 terminal-truncated mutants. HEK293A cells were transfected with expression vectors for FLAG-tagged ubiquitin (FLAG-Ubq, 4 µg), Myc-tagged PIAS1 mutants (3 µg), and necdin (1 µg), cultured for 20 hrs, and treated with 30 µM MG132 for 4 hrs prior to harvest. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody (Myc) and immunoblotted (IB) with anti-FLAG antibody. Expression levels of Myc-PIAS1 mutants (Myc), necdin (Necdin), FLAG-ubiquitin (FLAG) and γ-tubulin (Tub) were analyzed by immunoblotting (B–D).