Alexa fluor 488 conjugated phalloidin

Following 24 h-incubation, cells were fixed and stained as previously described. Cells were stained for F-actin (Alexa fluor 488-conjugated phalloidin, Invitrogen),DAPI (Sigma Aldrich UK) and cortactin (Anti-cortactin (p80/85) Antibody, clone 4F11; Millipore).

Cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 1% Triton-X. Cells were then immunostained with rabbit polyclonal cortactin primary antibody (H-191, Santa Cruz Biotechnology, Santa Cruz, CA) and Alexa Fluor 594 goat anti-rabbit secondary antibody (Invitrogen) or phospho-Src (Tyr416) Antibody (2101, Cell Signaling Technology, Danvers, MA) and Alexa Fluor 488 donkey anti-rabbit secondary antibody (Invitrogen). F-actin was stained with Alexa Fluor 488 conjugated phalloidin (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Images were taken on a LSM 700 confocal microscope (Zeiss, Germany) or an Axio Observer.Z1m microscope (Zeiss, Germany). The presence of podosomes was determined by co-localization (yellow) of F-actin (green) with cortactin (red), a common marker of podosomes [8 (link)].

The following reagents were purchased from the indicated sources: anti-α-tubulin antibody (clone DM1A), 12-O-tetradecanoyl-13-acetate (TPA), paclitaxel, and nocodazole (Sigma-Aldrich, St. Louis, MO, USA); 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Nacalai Tesque, Kyoto, Japan); Dynabeads^® Protein G, Alexa Fluor™ Plus 647-conjugated anti-mouse IgG antibody, Alexa Fluor™ Plus 555-conjugated anti-mouse IgG antibody, Alexa Fluor™ 488-conjugated phalloidin, and pHrodo™ Green Zymosan A BioParticles™ Conjugate (Invitrogen, Oslo, Norway); AcidiFluor™ ORANGE-NHS (AFO: Goryo Chemical, Hokkaido, Japan); formaldehyde (Polysciences, Warrington, PA, USA).

Embryos were methanol-heat-fixed (Miller et al. 1989 (link)) and stained with rabbit anti-Zip (1:200), rabbit anti-Anillin (1:100) (Goldbach et al. 2010 (link)), mouse anti-Nrt (1:10, Developmental Studies Hybridoma Bank, DSHB), and mouse anti-Arm-N27A1 (1:50, DSHB). Embryos were fixed in 4% formaldehyde/phosphate buffer with heptane (Karr and Alberts 1986 (link)), and stained with mouse anti-Dlg (1:20; DSHB), and mouse anti-Pnut (1:10; DSHB). Goat anti-mouse and anti-rabbit secondary antibodies were conjugated to Alexa Fluor 488, 546, or 680 (Invitrogen). Embryos were fixed in 8% formaldehyde/phosphate buffer with heptane (Warn and Magrath 1983 (link)), and stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen) to visualize F-actin. Embryos were mounted in Aquapolymount (Polysciences, Warrington, Pennsylvania), and imaged using an Olympus FluoView 300 or a Nikon Ti-E A1 confocal microscope. Image analyses were performed using ImageJ (Schneider et al. 2012 (link)). Circularity index (c = 4πA/p², where A = area, p = perimeter) was determined (Thomas and Wieschaus 2004 (link)), and tested using a two-sided Mann-Whitney test. Circularity index data from wild type and drak^del were compared to circularity index data from drak^del lines carrying phosphomimetic sqh transgenes, and nonphosphorylatable sqh transgenes, using a Kruskal-Wallis test with Dunn’s post-test.

Immunofluorescence analysis (IFA) was performed as described previously^{20 (link)}. Briefly, HeLa cells (2 × 10⁴ cells/well) grown on glass coverslips treated with or without specific pharmacological agents and CARDS toxin (140 pmol) or C-CARDS (70 pmol) were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and blocked with 1% normal goat serum (NGS; Gibco). Then, cells were washed with 0.2% NGS and treated with rabbit polyclonal anti-CARDS toxin (1:1000) as indicated previously and incubated with secondary goat polyclonal anti-rabbit antibody (1:1000 dilution) labeled with Alexa Fluor 555 (Invitrogen) for 1 h. Cellular F-Actin was stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen). For co-IFA studies, cells were incubated with GM130 (1:1000, Abcam) or ERGIC (1:100; Santa Cruz) monoclonal antibodies in PBS with 0.2% NGS in PBS for 1 h. Cells were washed with PBS containing 0.2% NGS and incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, 1:500) in PBS with 0.2% NGS for 1 h at RT. Individual samples were mounted in medium containing 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories;), and images were acquired using a Carl-Zeiss immunofluorescence microscope, and Z sections were prepared using AxioVision deconvolution software and enhanced in Adobe Photoshop.