Figure 1: Tctp physically interacts and colocalizes with Brm.

(a) Y2H (yeast two-hybrid) assay. A functional Gal4 formed by direct interaction between Tctp bait and Brm prey activates the expression of Gal4-responsive selection markers (URA3 and ADE2), allowing yeast transformants to grow on the selection media SD-LWU (-uracil) or SD-LWA (–adenine). (b) Reciprocal co-IP. Brm (2xFLAG-Brm^WT) is co-immunoprecipitated with Tctp (2xMYC-Tctp) in S2 cell and vice versa. (c–e) in vitro GST-Pull down assays. (c) Schematic domain structure of Brm and a list of mutated Brm polypeptide fragments used in pull-down assays. The full-length Brm protein has 6 conserved domains. (d) Direct Tctp binding to the middle part of Brm protein (304-747 amino-acid (aa) region, lane 4). (e) Both HSA and BRK domains of Brm are required for direct binding to Tctp (lane 4, 6, 7). (f) co-IP in S2 cell expressing 2xMYC-Tctp and 2xFLAG-full-length Brm with deletion of either HSA or BRK, or both domains. Deletion of either HSA or BRK domain of Brm reduces its interaction with Tctp (lanes 8, 9). Contrary to in vitro pull down assay, Brm^ΔHSA,ΔBRK shows a weak interaction with Tctp (lane 10). Tctp^E12V (E12V substitution) substantially interferes with the binding between Tctp and Brm (Lanes 2-5). (g–j) Tctp colocalizes with Brm in SG polytene chromosomes. (g) Squashed polytene chromosomes from the mixed SG tissues of wild-type and Tctp mutant larvae, stained with DAPI (blue), Tctp (red), and Brm antibodies (green). Scale bar, 50 μm. Contrary to wild-type chromosomes (red, arrowhead), Tctp^EY/h59mutant chromosomes show reduced staining with anti-Tctp antibody (arrow). (h) Enlarged split images of the two boxed regions of wild-type chromosomes shown in g. Most Tctp signals are colocalized with Brm (yellow arrowheads), but some areas show preferentially either Tctp (red arrowheads) or Brm (green arrowheads). (i) The quantified gross bands intensities of Tctp and Brm staining normalized to DAPI (n=10 SGs, mean±s.d.). NS, not significant (P=0.8).