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. 2014 Apr;20(4):568–579. doi: 10.1261/rna.043513.113

FIGURE 4.

FIGURE 4.

(A) Far-Western dot-blot assay investigating the interaction between the recombinant AnaRneC and AnaPnp purified from E. coli cells with or without the treatment with micrococcal nuclease (MNase). AnaPnp and BSA were dotted onto the membranes as a positive control and a negative control, respectively. (B) Determining the complex of AnaRneC and AnaPnp by gel electrophoresis. AnaRneC and AnaPnp were incubated under native conditions for 30 min, then the mixture was separated on the native-PAGE gel (the middle lane in the left panel). The band of protein complex (indicated by an arrow) in the native gel was excised and subsequently subjected to SDS-PAGE electrophoresis (the middle lane in the right panel). Purified AnaRneC and AnaPnp were included as size markers in both the native-PAGE gel and the SDS-PAGE gel. Note that AnaRneC did not migrate as a sharp band in the native-PAGE gel. (C) Bacterial two-hybrid assay investigating the interaction between AnaRneC and AnaPnp. The E. coli XL1-Blue MRF′ Kan cells cotransformed by the indicated two-hybrid plasmid pairs were spotted on the selective and the nonselective plates, respectively. Cells cotransformed with pBT-LGF2 and pTRG-Gal11P (supplied in the kit) were used as the positive control (CK+), and cells cotransformed with the empty vectors of pBT and pTRG were used as the negative control. Note that, due to unclear reasons, cells containing pTRG-AnaPnp/pBT or pTRG-AnaPnp/pBT showed weak growth.