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

Figure 4. Necdin promotes PIAS1 degradation via the ubiquitin-proteasome pathway.

Figure 4

(A) Necdin-dependent PIAS1 degradation. HEK293A cells were transfected with expression vectors for Myc-tagged PIAS1 (Myc-PIAS1, 3 µg) and necdin (0.5, 1, 2 µg). (B) Proteasome-mediated degradation of PIAS1. HEK293A cells were transfected with expression vectors for Myc-PIAS1 (3 µg) and necdin (1 µg), incubated for 20 hrs, treated with 30 µM MG132 (MG132+) or DMSO (MG132-) for 4 hrs, and harvested. Expression of Myc-PIAS1 (Myc), necdin and γ-tubulin (Tub) was analyzed by immunoblotting (A, B). (C, D) Necdin-promoted PIAS1 ubiquitination. HEK293A cells were transfected with combinations of expression vectors for FLAG-tagged ubiquitin (FLAG-Ubq, 4 µg)(C), HA-tagged ubiquitin (HA-Ubq, µg)(D), Myc-PIAS1 (3 µg), necdin (1 µg) (C) and FLAG-tagged necdin (ND, 2 µg), MAGEA1 (A1, 2 µg), and necdin-like 2 (NL, 1 µg) (D). Transfected cells were incubated for 20 hrs, treated with 30 µM MG132 for 4 hrs, and harvested. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody (Myc) and immunoblotted (IB) with antibodies to FLAG (C) and HA (D). Expression of Myc-PIAS1 (Myc), necdin, FLAG-tagged proteins (FLAG), HA-Ubiquitin (HA) and γ-tubulin (Tub) was analyzed by immunoblotting. Abbreviations (D): ND, necdin; A1, MAGEA1; NL, necdin-like 2.