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

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

The interaction of su(Hw) with Cp190 is RNA-independent (not affected by RNaseA treatment).

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

Positive control.

Two-hybrid system: yeast

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

Cp190 does not bind DNA on its own; when mixed with su(Hw), a supershift forms. Addition of mod(mdg4) results in a more pronounced supershift.

su(Hw) and Cp190 were mixed with [32]P labeled DNA. DNA-protein complexes observed by EMSA were used to deduce protein-protein interaction.

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

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.

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 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.

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 cell extract of S2 cell line; bait produced from transfected construct; prey produced from endogenous gene.

TAP tandem affinity purification of bait and identification of prey by mass spectrometry.

su(Hw) enriched 6-fold in Cp190 purification compared to negative control.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from endogenous gene.

Source was cell extract of S2R+ cell line; proteins produced from transfected construct or endogenous gene.

HGScore = 34.521684

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

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).

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

Two-hybrid system: yeast GAL4-BD/GAL4-AD. An H to Y mutation in zinc finger 10 of su(Hw) disrupts the su(Hw)-Cp190 interaction while deletion of zinc finger 10 has no effect suggesting the H to Y mutation affects su(Hw) conformation.

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

An H to Y mutation in zinc finger 10 of su(Hw) disrupts the su(Hw)-Cp190 interaction while deletion of zinc finger 10 has no effect suggesting the H to Y mutation affects su(Hw) conformation.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from endogenous gene.

An H to Y mutation in zinc finger 10 of su(Hw) disrupts the su(Hw)-Cp190 interaction while deletion of zinc finger 10 has no effect suggesting the H to Y mutation affects su(Hw) conformation.

Source was adult males of transgenic fly line; bait produced from tagged transgenic construct; 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

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 from cDNA library of 154 Drosophila C2H2 domain proteins).

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

Cp190 interacts with su(Hw) bound to DNA.

su(Hw) and Cp190 was mixed with Cy5 labeled DNA. DNA-protein complexes observed by EMSA were used to deduce protein-protein interactions.

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

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

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).