Goat anti mouse dylight 488

24 h post-transfection, HEK293 cells overexpressing wildtype and mutant V5-PFN1 were washed in PBS, fixed for 10 min in Roti-Histofix 4 % (Roth), permeabilized for 10 min in PBS supplemented with 0.1 % Triton X-100 and 100 mM glycine and blocked for 45 min in PBS with 1.5 % bovine serum albumin and 0.1 % Tween20. Afterwards, cells were incubated for 1 h with the primary antibody mouse anti-V5 (see above; 1:750 in PBS), washed in PBS and incubated for 1 h with the secondary antibody goat anti-mouse DyLight 488 (Thermo Fisher Scientific Cat# 35502 RRID:AB_844397; 1:500 in PBS) and Phalloidin-Atto 594 (Sigma-Aldrich Cat# 51927; 600 nM in PBS) followed by washing in PBS. Cell nuclei were stained for 10 min using DAPI (1 µg/ml in PBS). After a final washing in PBS, cells were mounted on microscope slides and analyzed using a Carl Zeiss Axio Observer.A1 microscope.

Seeds of the 35S:H3.3-YFP line were germinated and grown for 3 days in Petri dishes on wet filter paper. For visualizing nuclear DNA in live cells, 1 μM of DRAQ5 (eBioscience, Vienna, Austria) was applied to Arabidopsis roots for 5 to 10 minutes with vacuum to facilitate penetration. DRAQ5 stain and YFP signals in roots were consecutively analyzed using a Zeiss 710 confocal laser scanning microscope.
For immuno-staining, seedlings were fixed for 20 minutes with ice-cold 4% (w/v) paraformaldehyde in MTSB buffer (50 mM PIPES, 5 mM MgSO₄, 5 mM EGTA, pH 6.9). Root tips were digested for 10 minutes at 37°C with a PCP enzyme mixture (2.5% pectinase, 2.5% cellulase Onozuka R-10, 2.5% Pectolyase Y-23 (w/v) dissolved in MTSB) and squashed in a drop of MTSB buffer. Immunostaining was performed as described
[58 (link)]. H3.3-YFP was detected with rabbit anti-GFP (1:100; #A11122, Molecular Probes, Eugene, OR, USA) and donkey anti-rabbit Rhodamine (1:200; #31685, ThermoScientific, Waltham, MA, USA). Pol II was detected using mouse anti-Pol II (1:100; #ab817, Abcam, Cambridge, England) and goat anti-mouse Dylight488 (1:200; #35503, ThermoScientific). For confocal laser scanning microscopy, 35S:H3.3-YFP seedlings were grown on Murashige and Skoog medium for 5 days before YFP signals in roots were analyzed using a Zeiss 710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

The following antibodies were used in this study: anti-EGFR (Cell Signaling Technology, Danvers, MA), anti-STAT3 and anti-vimentin (Proteintech Group Inc, Chicago, IL), anti-phospho-EGFR (pY1173) and anti-phospho-STAT3 (pY705) (Epitomics, Burlingame, CA), anti-CD44, anti-SNAI1 and anti-α-tubulin (Santa Cruz Biotechnology, Dallas, TX), anti-N-cadherin, goat anti-mouse DyLight 488 and goat anti-rabbit DyLight 488 (Thermo Fisher Scientific, Rockford, IL), PE-conjugated anti-human CD147 and isotype-matched mouse immunoglobulin (Miltenyi Biotec, Auburn, CA), and goat anti-rabbit Texas-Red and goat anti-mouse fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch, West Grove, PA). Goat anti-rabbit horseradish peroxidase (HRP), goat anti-mouse HRP, mouse IgG, geneticin (G418) and Lipofectamine 2000 transfection reagent were purchased from Invitrogen (Carlsbad, CA). The anti-mouse HAb18G/CD147 antibody HAb18IgG was prepared as previously reported [16 (link)].
Recombinant human CyPA was purchased from Sigma-Aldrich (St. Louis, MO), puromycin was purchased from InvivoGen (San Diego, CA), WP1066 was obtained from Calbiochem (Billerica, MA), gemcitabine was purchased from Lilly France S.A. (Fegersheim, France), and PD153035 and gefitinib were obtained from Selleck (Boston, MA).