Rat anti ci 2a1

Immunostaining of imaginal discs was carried out as previously described (Jiang & Struhl, 1995 (link)). Immunofluorescence imaging was performed using a Zeiss 710 laser scanning microscope. For immunoprecipitation assay, after transfection for 48 h, cells were washed twice with PBS and then lysed on ice for 10 min with lysis buffer containing 1M Tris, pH 8.0, 5M NaCl, 1MNaF, 0.1M Na3VO4, 1% CA630, 10% glycerol, and 0.5M EDTA (pH 8.0). Cell lysates were incubated with protein A-Sepharose beads (Thermo Fisher Scientific) for 1 h at 4°C to eliminate nonspecific binding proteins. After removal of the protein-A beads by centrifugation, the cleared lysates were incubated with Myc (HA or Flag) antibody for 2 h or overnight. Protein complexes were collected by incubation with protein A-Sepharose beads for 1 h at 4°C, followed by centrifugation. Immunoprecipitates were washed three times for 5 min each with lysis buffer and were separated on SDS–PAGE. Western blot was carried out using standard protocol. Antibodies used in this study were as follows: mouse anti-Myc (Santa Cruz Biotechnology), mouse anti-Flag (Sigma-Aldrich), mouse anti-HA (Santa Cruz Biotechnology), rat anti-Ci 2A1, and mouse anti-Ptc (Developmental Studies Hybridoma Bank).