Effects of CD44 ligation on the expression of cell cycle regulatory molecules in NB4 cells. (A) NB4 cells were cultured with A3D8 as described in “Materials and methods.” After western blotting with specific Abs, the protein levels of Cdk2, Cdk4, and Cdk6; cyclins D1 and E; CKI p27 were evaluated by densitometric scanning, corrected with respect to α-tubulin expression, and expressed relative to the value obtained at time 0 (arbitrarily 1). These relative protein level values are shown in italics below the lanes. Results represent 1 representative experiment of 3. (B) Confocal microscopic analysis of p27 localization in A3D8-treated and control (IgG1-treated) NB4 cells: Cells were permeabilized and successively incubated with a polyclonal rabbit anti-p27 Ab and a goat antirabbit Ab conjugated with Alexa-488 dye (green color). The nucleus was stained with propidium iodide (red color). The yellow color (in A3D8-treated cells) results from the superposition of green and red staining and reveals the nuclear localization of p27. Results represent 1 representative experiment of 2.