Fig. 3. CD81 resides in a complex with v5 but not with MerTK or CD36 in RPE cells. (A) Polarized RPE-J cells were separated into extracted (Ex) and insoluble pellet (P) fractions by differential extraction with buffer containing 1% Triton X-100 (Tx), Brij97 (B97), or CHAPS (Ch) either still on ice or with vortexing as indicated. Equal volumes of fractions were analyzed by western blotting with antibodies as indicated to the right of the panels. (B) IPs from vortexed RPE-J Brij97 lysates (as in A) with antibodies to CD81 or phagocytic receptors 5 integrin, MerTK and CD36, and non-immune hamster and goat antibodies were probed sequentially in the order of panels shown (top to bottom) for precipitated receptors and for CD9 as indicated to the left of each panel. CD81 and 5 antibodies co-isolated CD81, CD9, v, and 5. Because after sequential re-probing of blots ezrin antibody showed only very weak ezrin signals, receptor IPs were repeated and fresh blots probed with ezrin yielding robust bands representing co-precipitated ezrin specifically with CD81 and 5 integrin (panel ezrin). (C) Tissue samples of neural retina (NR) and corresponding RPE-choroid (RPE/Ch) isolated from individual 5^+/+ and 5^–/– mouse eyes were lysed in buffer containing Brij97. IPs from these tissue lysates with CD81 antibody co-precipitated v integrin from 5^+/+ neural retina and RPE/choroid and from 5^–/– neural retina but not from 5^–/– RPE/choroid (RPE/Ch) (v panel). CD81 immunoblotting revealed that CD81 antibody precipitated similar amounts of CD81 from 5^+/+ and 5^–/– tissues (CD81 panel). Non-immune antibodies did not isolate any of the proteins of interest (data not shown).