Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

Crosslinked Cp190 ran as a dimer.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

Cp190 eluted as a tetramer.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

Cp190 ran as a tetramer.

Extracts were sequentially passed over anti-su(Hw) and anti-Cp190 columns. Associated RNA was 5\' end labeled and gel purified and characterized by next generation sequencing. Alternatively, associated RNA was isolated by oligo-dT selection for polyadenylated transcripts.

Using dl as a negative control, it was shown that a chromatin-associated protein does not typically copurify with its own coding transcript in this protocol.

Using gel purification to isolate RNA copurifying with insulator proteins su(Hw) and Cp190, only su(Hw) and Cp190 transcripts were significantly enriched. The RNA was 35-55 nt in size and mapped to the sense strand within exons and across exon junctions throughout the entirety of the gene (likely degradation products of full-length mRNA). su(Hw) and Cp190 transcripts were also identified in RNA isolated by oligo-dT selection.

Source was embryonic nuclear extract of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast GAL4-BD/GAL4-AD; positive control

Two-hybrid system: yeast GAL4-BD/VP16-AD

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

MW values were determined from column calibration with standard proteins. Cp190 forms a homodimer.

Interaction in vitro; protein produced as a recombinant fusion proteins in bacterial system.