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. 2016 May 2;44(15):7228–7241. doi: 10.1093/nar/gkw371

Figure 2.

Figure 2.

Testing for homo- and heterodimerization of the ZAD domains in vitro. (A) Sequence comparison of ZAD domains of Grau, Pita, ZIPIC and Zw5. (B) Testing for homodimerization of Grau, Pita, ZIPIC, and Zw5 ZAD domains by chemical crosslinking with glutaraldehyde. (C) The results of MBP and 6×His pull-down assay showing that the ZAD domains of proteins expressed in vitro formed only homodimers. For MBP pull-down, beads with bound MBP–Pita, MBP–ZIPIC, MBP–Zw5 or MBP alone were incubated with 6×His-Trx-Pita, 6×His-Trx-ZIPIC, 6×His-Trx-Zw5, 6×His-Trx-Grau or 6×His-Trx. For 6×His pull-down, beads with bound 6×His-Trx-Pita, 6×His-Trx-ZIPIC, 6×His-Trx-Zw5, 6×His-Trx-Grau, were incubated with MBP–Pita, MBP–ZIPIC, MBP–Zw5 or MBP alone. The precipitated proteins were resolved by SDS-PAGE and stained with Coomassie. Grau was used as positive control for homodimer formation, while MBP and 6×His-Trx were used as negative control. Arrows indicate the precipitated prey protein after pull-down with bait protein.