Computational protocol: Sticky/Citron kinase maintains proper RhoA localization at the cleavage site during cytokinesis

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Protocol publication

[…] The following antibodies were used in this study: mouse monoclonal anti–α-tubulin (clone DM1A; Sigma-Aldrich), chicken polyclonal anti–α-tubulin (ab89984; Abcam), mouse monoclonal antiactin (clone AC-40; Sigma-Aldrich), mouse monoclonal anti-Myc (clone 9E10; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Myc (ab9106; Abcam), rabbit polyclonal anti-Sti (), mouse monoclonal anti-Rho1 (clone p1D9; Developmental Studies Hybridoma Bank), rat anti-Sqh2P and guinea pig anti-Sqh1P (provided by R. Ward, University of Kansas, Lawrence, KS; ), rabbit polyclonal anti-MRLC S19 (Cell Signaling Technology), peroxidase-ChromPure anti–rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.), and Alexa Fluor 594 phalloidin (Invitrogen). Peroxidase- and Alexa Fluor–conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. and Invitrogen.Dmel cells were grown on 22 × 22–mm coverslips (Menzel-Gläser) and fixed in PHEM buffer (60 mM Pipes, 25 mM Hepes, pH 7, 10 mM EGTA, 4 mM MgCl2, and 3.7% formaldehyde) for 12 min. They were then washed three times for 10 min with PBS and incubated in blocking buffer (PBS, 0.5% Triton X-100, and 3–5% BSA) for 1 h at room temperature. Incubation with primary antibodies was performed overnight at 4°C in PBT (PBS, 0.1% Triton X-100, and 1% BSA). After two washes in PBT, coverslips were incubated with Alexa Fluor–conjugated secondary antibodies (Invitrogen) diluted in PBT for 2 h at room temperature and then washed two times with PBT and once with PBS. Coverslips were finally mounted using mounting medium with DAPI (Vectashield; Vector Laboratories) and visualized on a fluorescence microscope (Axiovert 200; Carl Zeiss) equipped with a 100× objective, NA 1.4. Images were acquired using a camera (CoolSNAP HQ2; Photometrics) with MetaMorph software (Molecular Devices) and assembled and adjusted for contrast using Photoshop CS3. Quantification of Sqh fluorescence was performed using ImageJ software (National Institutes of Health), and fluorescence intensity values were plotted using Excel software (Microsoft). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Drosophila melanogaster
Chemicals Guanosine, Potassium