Fig. 2. Characterization of anti-Alfy antibodies. (A-D) HeLa cells were transfected with Myc-Alfy2587-3527 and analysed by western blotting (A) or immunofluorescence microscopy with anti-Myc (A,B) and anti-Alfy (A,C) antibodies. (E) The specificity of the anti-Alfy antibody was analysed using affinity-purified anti-Alfy serum or serum depleted of anti-Alfy antibody for immunoblotting of a gel containing cytosol (C) or membrane (M) fractions from HeLa cells. (F) HeLa cells were incubated with an Alfy-specific siRNA, a scrambled siRNA or transfection reagent alone. Equal amounts (60 µg protein) of cell lysates were analysed by western blotting with affinity-purified anti-Alfy antibodies.