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. 2014 Jan 28;26(1):454–464. doi: 10.1105/tpc.113.120311

Figure 2.

Figure 2.

UBC24 (PHO2) Is a Specific E2 Conjugase for NLA.

(A) In vitro pull-down assay of His-UBC8 with MBP-NLA full-length. His-UBC8 as prey was mixed with MBP or MBP-NLA as bait. His-UBC8 was analyzed by protein gel blot using anti-His antibody.

(B) In vitro pull-down assay of GST-UBC24 with MBP-NLA full-length. GST-UBC24 as prey was mixed with MBP or MBP-NLA as bait. GST-UBC24 was analyzed by protein gel blot using anti-GST antibody.

(C) UBC8 does not support NLA self-ubiquitination.

(D) UBC24 supports NLA autoubiquitination.

For (C) and (D), the reaction mix contained 25 ng UBE1 (rabbit E1), 100 ng UBC8 or UBC24 (E2), 500 ng MBP-NLA (E3), and 8 μg His-Ubiquitin. After the reaction, polyubiquitinated NLA was analyzed by protein gel blot using anti-MBP antibody.