Fig. 2. ALCAM is associated with the actin cytoskeleton. (A) Surface expression of ALCAM on K562, K562-ALCAM, KG1a and undifferentiated MUTZ-3 cells was analyzed by flow cytometry. Unfilled histograms represent isotype control staining and shaded histograms represent staining with ALCAM antibody, AZN-L50. The mean fluorescence intensity (MFI) and percentage of positive cells are as indicated. (B) A 42 kDa protein co-precipitates with ALCAM from KG1a cells, as indicated by the arrows. KG1a and undifferentiated Mutz-3 control cells (no ALCAM expression) were incubated overnight with [³⁵S]methionine/cysteine. ALCAM was immunoprecipitated from labeled cell lysates with 1 µg of AZN-L51. Samples that were incubated with protein G beads alone (–) are shown as negative controls. (C) Identification of the co-precipitated protein by western blot analysis. ALCAM was immunoprecipitated from labeled KG1a cell lysates with 1 µg of AZN-L51. ALCAM and β-actin were detected using antibodies AZN-L50 and anti-β-actin (clone AC-15), respectively. As a negative control, lysates were incubated with an irrelevant control antibody (anti-hemagglutinin, clone 12CA5) or with protein G beads alone (–).