Figure 3.

UBC24 E2 Conjugase Is Required for PT2 Polyubiquitination by NLA E3 Ligase.

(A) and (C) In vitro pull-down assay of PT1 (C) and PT2 (A) with NLA. MBP or MBP-NLA as bait and GST-PT1 or GST-PT2 as prey were mixed. GST-PT1 or GST-PT2 was analyzed by protein gel blots using anti-GST antibody.

(B) and (D) In vitro ubiquitination of PT1 (D) and PT2 (B). The reaction mix contained 25 ng UBE1 (rabbit E1), 100 ng UBC8 or UBC24 (E2), 500 ng MBP-NLA (E3), 100 ng of substrate (GST-PT1-MYC or GST-PT2-MYC), and 8 μg His-Ubiquitin.

After the reaction, polyubiquitinated GST-PT1-MYC or GST-PT2-MYC was analyzed by protein gel blot using anti-MYC antibody.