Mouse anti profilin

For antibody staining, hemocytes were bled from third-instar larva and allowed to attach to a glass slide for 30 min. The cells were then fixed at room temperature with 3.7% formaldehyde in PBS for 10 min, pre-incubated in blocking solution (PBS with 0.1% Tween-20 and 5% goat serum) and incubated with primary antiserum diluted in blocking solution. The following primary antibodies were used: mouse anti-NimC1, mouse anti-L1 and mouse anti-H2 (gifts from I. Ando); rat anti-Jumu (made in our lab); mouse anti-α-tubulin (sigma); rabbit anti-PH3 (Millipore); mouse anti-Dorsal, mouse anti-Ena, mouse anti-Fascin, mouse anti-Rho1 and mouse anti-Profilin (Developmental Studies Hybridoma Bank); rabbit anti-Dif (gift from Dominique Ferrandon). Alexa Fluor 488-, Alexa Fluor 568- and Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) were employed. For phalloidin staining, hemocytes were preincubated with PBST (PBS with 0.1% TritonX-100) for 5 min and then incubated with Alexa Fluor 488-labeled phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. Images were obtained using a Zeiss Axioplan 2 microscope equipped with fluorescence optics. All staining was performed in at least three independent experiments.

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.