Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis

Figure 2

(a) Slot blot analysis of binding capacity of CTA 157-2 with different concentrations of SDS, CHAPS, and Triton. (b) Confirmation of purity of membrane fractions with anti-CD 54 antibody and cytosolic fractions with anti-PKG antibody. (c) Solubility of VR-EPC membrane fraction in different detergents (S: soluble phase; P: precipitate). (d) CTA 157-2 staining of different fractions collected after gel chromatography of cell membrane preparations along with stained marker proteins of defined sizes reveals that CTA 157-2 stains a protein complex of 700 kDa. (e) This complex separates on conventional SDS page into several subunits identified as 26 kD α7 and 21 kD β3 proteasome in mass spectrometry. (f) Specific staining of 20S proteasome after immunoprecipitation with CTA 157-2 of VR-EPC extracts (antigen positive cells). No staining after immunoprecipitation of antigen negative rat heart microvascular cells (antigen negative cells). No staining after immunoprecipitation with IgM control and of CTA 157-2 sepharose beads.

(a)

(b)

(c)

(d)

(e)

(f)