Cells were washed with PBS and fixed for 20 min with 2% paraformaldehyde in PBS. Cells were permeabilized for 5 min with PBS/0.2% Triton X-100 and blocked with PBS/0.2% Tween 20 containing 5% bovine serum albumin. Coverslips were incubated for 1 hour with primary antibodies and for 30 min with appropriate secondary antibodies coupled to Alexa Fluor 488, 594, or 647 fluorophores (1:1000; Life Technologies) before being incubated with DAPI (2 μg/ml). Pictures were acquired with a FluoView 1000 confocal microscope (Olympus) using the same laser power for matching images and antibodies. All the immunofluorescence experiments were performed at least three times independently, and the pictures shown in the figures are representative images of the three experiments. Image analyses were carried out with Volocity software (PerkinElmer) using the following protocol: find objects (DAPI); subpopulation, fill holes in objects; exclude touching edge of image; exclude objects by size, <100 μm2; measure. The fluorescence intensity in each channel was then recorded.

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