The effect of detergents on CD36-associated proteins.
(A) Platelet membrane proteins were labeled with biotin and lysed in 1% CHAPS (lane 1), Triton X-100 (lane 2), Brij 96 (lane 3), or Brij 99 (lane 4), and CD36 was immunoprecipitated with the monoclonal antibody FA6-152. In Triton X-100 platelet lysates (lane 2), only CD36 is precipitated, whereas in CHAPS (lane 1), Brij 96 (lane 3), and Brij 99 (lane 4) platelet lysates, additional protein bands can be coprecipitated. (B) Two other anti-CD36 monoclonal antibodies 1E8 (lane 1) and 185-1G2 (lane 2) also precipitate the complex in Brij 99 lysates, but a nonrelated antibody to CD31 (lane 3) and nonimmune mouse IgG (lane 4) do not. Lane 5 is the complete platelet lysate, showing that multiple membrane proteins are biotinylated and the bands that are immunoprecipitated with anti-CD36 antibody are not selectively labeled. (C) The distribution of CD36 in the supernatant (S) or pellet (P) after solubilization in the various detergents. Platelets (5 × 108) were solubilized in 1.0 mL lysis buffer that contained 1% of the various detergents. After 20 minutes at 4°C, the sample was centrifuged, and the supernatant was removed. The pellet was resuspended in one half the original volume of SDS-PAGE sample buffer and 10 μL supernatant or 5 μL solubilized pellet was subjected to Western blot analysis with an anti-CD36 antibody.
