Fig. 4: Mobile α_vβ₃ integrin and CD47 appear confined in the same membrane area.

a CD47 was detected after β3 integrin subunit immunoprecipitation from a HeLa cell lysate. The cell lysate was absorbed to non-coated magnetic beads (Control) or coated with anti-integrin β3 (AB2984). CD47 was next detected by western blotting with an anti-CD47 antibody (B6H12). As indicated, CD47 detected on a whole cell lysate (total cell lysate) and CD47 detected on the β3 integrin subunit-immunoprecitated sample (co-immunoprecipitation). Samples were submitted to 10 % SDS PAGE analysis under non-reducing conditions. The position of CD47 is indicated by a (*). A non-specific band is detected at 34 kDa in both samples. Molecular weight markers (kDa) are shown. b Two color single particle tracking of integrin α_Vβ₃ and CD47 was obtained using anti-αVβ3 (23C6) and anti-CD47 (B6H12) antibodies labeled with PE and A647 respectively, and imaged by TIRF microscopy during 120 s at 33 Hz. The localizations of the two proteins were compiled over time to reconstruct super-resolved images. White dotted squares numbered 1, 2, 3 delimit enlarged zones on the right and yellow dotted rectangles delimit zones used to calculate Manders’ coefficients. M^αvβ3 and M^CD47, Manders’ overlap coefficient of αvβ3 localization with CD47 and of CD47 with αvβ3, respectively. Scale bar, 5 µm. c MJD distribution for αvβ3 extracted from SPT acquisition as previously done in Figs. 2 and 3. Dotted red lines correspond to the CD47 mean MJD value of the immobile population extracted from formaldehyde + glutaraldehyde fixation (44 nm), and from the low mobility (111.9 nm) and high mobility (172.7 nm) population extracted from control viable cells. The MJD distribution was obtained after merging MJDs from several cells (n = 12) from two independent experiments.