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. 2012 Jun 4;159(4):1355–1366. doi: 10.1104/pp.112.199356

Figure 6.

Figure 6.

Interactions between XXTs confirmed by in vitro pull-down assay. A, In vitro Ni-NTA pull-down assay for interaction between tXXT2His and tXXT5Myc. tXXT2His and tXXT5Myc lysates and a mixture of the two lysates were applied to Ni-NTA affinity columns. The elution fractions were detected by either anti-His or anti-Myc antibody (Ab). Asterisks indicate nonspecific bound signal. B, In vitro Ni-NTA pull-down assay for the interaction of tXXT1HA and tXXT2His. tXXT1HA and tXXT2His lysate mixture was applied onto a Ni-NTA affinity column. The elution fractions were detected by either anti-His or anti-HA antibody. Asterisks indicate nonspecific bound signal. C, In vitro Ni-NTA pull-down assay for the interaction of tXXT1HA and tXXT5His. tXXT1HA and tXXT5His lysate mixture was applied onto a Ni-NTA affinity column. The elution fractions were detected by either anti-His or anti-HA antibody. The flow-through fraction was detected by anti-HA antibodies to confirm the presence of nonbound tXXT1HA. D, In vitro Ni-NTA pull-down assay for interaction between tXXT2His and tXXT2HA. tXXT2His and tXXT2HA lysate mixture was applied onto a Ni-NTA affinity column. The elution fractions were detected by either anti-His or anti-HA antibody. E, In vitro Ni-NTA pull-down assay for interactions between tXXT5His and tXXT5Myc. tXXT5His and tXXT5Myc lysate mixture was applied onto a Ni-NTA affinity column. The elution fractions were detected by either anti-His or anti-Myc antibody. F, In vitro Ni-NTA pull-down assay for interactions between tXXT1His and tXXT1HA. tXXT1His and tXXT1HA lysate mixture was applied onto a Ni-NTA affinity column. The elution fractions were detected by either anti-His or anti-HA antibody. The flow through fraction was detected by anti-HA antibodies to confirm the presence of nonbound tXXT1HA. G, In vitro Ni-NTA and anti-HA agarose pull-down assay for interactions between tXXT2His and HA tag-fused full-length XXT5. tXXT2His lysate was mixed with protein extract from XXT5HA-expressing transgenic plants. The mixture was pulled down by either a Ni-NTA or HA agarose column. Both elution fractions were detected by anti-HA and anti-His antibodies.