Figure 4
Interaction of intracellular salusin-β with a membrane-associated β-actin-profilin complex. (a) Whole cell lysates obtained from the human cell lines HeLa, NB-1, THP-1 and U937 were immunoprecipitated with anti-profilin1/2 or anti-β-actin IgG and immunoblotted with an anti-salusin-β antibody. The upper and lower panels represent 3- and 30-min exposures, respectively, of the same immunoblot. (b) Whole cell lysates were immunoprecipitated with anti-profilin1/2 IgG and immunoblotted with the anti-salusin-β antibody (left panel), or immunoprecipitated with anti-PIP2 IgG and immunoblotted with anti-profilin1/2 IgG. (c) Immunofluorescence staining of NB-1 cells with the anti-salusin-β antibody reveals marked staining in the cell periphery. (d) Quiescent HeLa and THP-1 cells stimulated by 10% fetal bovine serum and endothelin-1 (10−9 M), respectively (upper and middle panels) and growing THP-1 cells stimulated by TNF-α (100 ng/ml, lower panel) for the indicated times were fractionated by differential centrifugation. Plasma membrane (PM)-, low-density microsome (LDM)-, mitochondria/nucleotide (M/N)-, and high-density microsome (HDM)-enriched fractions were immunoprecipitated with anti-profilin1/2 IgG, separated by SDS-PAGE and analyzed by immunoblotting with the anti-salusin-β antibody.
