Anti tra98

Cells were washed with PBS, attached to poly-L-lysine slides and fixed in 1% paraformaldehyde followed by washing twice in PBS. Fixed cells on slides were treated with 0.3% Igepal CA-630 for 10 min and blocked with Image-iT^® FX Signal Enhancer (Life technologies, Cat# I36933) for 30 min. Cells were then rinsed with PBS and incubated for 2 h with the following primary antibodies diluted in PBS containing 1% BSA: anti-CDK14 (ProteinTech, at 1:150 dilution); anti-CDK14 (Abcam, at 1:50 dilution); anti-GATA4 (Affymetrix eBioscience, Cat# 14-9980-80, at 1:200 dilution); anti-TRA98 (Abcam Cat# ab82527 at 1:100 dilution). Following one wash with PBS, cells were incubated with Alexa Fluor 488- or 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 594-conjugated or 488-conjugated goat anti-rat IgG (Life technologies) at a 1:100 dilution in PBS containing 1% BSA for 1 h. Following two washes, the nuclei were stained for 5 min with 4μg/mL of 4,6-diamino-2-phenylindole (DAPI) (Sigma–Aldrich, Cat# D9542). Slides were rinsed and mounted with ProLong^® Gold antifade reagent (Life technologies, Cat# 36930). Images were collected with a Nikon inverted fluorescence microscope using 60× and 100× objective lenses with DAPI, fluorescein isothiocyanate (FITC) and CY-5 filter sets. At least 50 cells were analyzed for each slide.