Cvcl z232

A total of 5 × 10⁵ S2 cells (CVCL_Z232, Invitrogen, Cat#R690-07) stably expressing the pMK33-Flag-jumu full CDS construct or empty pMK33 vector were plated per well in a 24-well plate and then co-transfected with 200 ng of dmyc promoter firefly luciferase reporter plasmid and 20 ng of Renilla luciferase pRL-TK reporter vector. The cells were harvested after 48 hr of incubation. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega), and the normalized luciferase values were calculated as the ratio of the firefly to the Renilla luciferase activities. Plasmid transfection was performed in at least triplicate, and luciferase activity was measured in duplicate or triplicate.

S2 cells (CVCL_Z232, Invitrogen, Cat#R690–07) were transfected with pMK33-Flag or the pMK33-Flag-jumu full CDS construct using the Effectene Transfection kit (Qiagen). Whole-cell extracts were prepared in lysis buffer containing 20 mM Tris (pH 7.6), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol (DTT), 2 mM EDTA, and protease inhibitors. Then, 30 μg of the lysate was loaded into a 12% SDS-PAGE gel, followed by electroblotting onto nitrocellulose membranes and probing with mouse anti-α-tubulin (1:500, Sigma), mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (1:300, Developmental Studies Hybridoma Bank) for 2 h. The blot was subsequently probed with anti-mouse HRP-conjugated secondary antibodies for 1.5 h and detected using the ECL Plus detection system (Program). ImageJ was employed to measure the intensity values of the blots. Representative blots obtained from at least three independent experiments with similar results are presented.

ChIP assays were performed as described previously (Kim et al., 2005 (link)). In brief, S2 cells (CVCL_Z232, Invitrogen, Cat#R690-07) were transfected with the pMK33-Flag-jumu full CDS construct using the Effectene Transfection kit (Qiagen). S2 cells stably expressing Flag-jumu were fixed in 1% formaldehyde at room temperature. The samples were then sonicated to obtain DNA fragments between 200 and 800 bp in length. The fragmented chromatin was incubated with the anti-Flag antibody (Sigma) or IgG (Santa Cruz) bound to Salmon Sperm DNA/Protein A Agarose Slurry (Upstate) overnight at 4°C. The immunoprecipitated DNA fragments were recovered using a MiniElute purification kit (Qiagen) and analyzed by PCR amplification. Similar results were obtained in three independent ChIP experiments. The primer sequences are shown in Supplementary file 1.