fig4

Figure 4. Immunomagnetic bead isolation confirms that RANK is mostly in EVs containing CD9. Conditioned media from osteoclasts from two wells of a 6-well plate for each trial were centrifuged at 5,000 × g for 15 min to remove cells and large debris. The conditioned media was subjected to magnetic immunoaffinity isolation. The total conditioned media, the unbound material and bound material eluted with low pH were subjected to SDS-PAGE, blotted to Immobilon P, and probed with antibodies against RANK, CD9 or CD81 as indicated. (A) Affinity isolation with anti-CD9 shows most of RANK was isolated with CD9-containing EVs; (B) Control shows neither RANK nor CD9 was isolated with control antibody; (C) Little RANK was isolated in CD81 EVs; (D) Little binding of RANK or CD81 was detected using a control antibody for anti-CD81; (E) Blots (n = 3) of CD9 immunomagnetic isolations were quantitated by densitometry. Most RANK and CD9 were isolated with anti-CD9 immunomagnetic isolation; (F) Blots (n = 3) of CD81 immunomagnetic isolations were quantitated by densitometry. A small but significant amount of RANK was detected associated with CD81. Note that the anti-CD81 antibody worked much better for the detection of its target protein in Westerns than the anti-CD9 antibody. Asterisks indicate a difference from control value; P < 0.05 by t-test; *: P < 0.05.