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. 2017 May 22;7:2246. doi: 10.1038/s41598-017-02356-1

Figure 1.

Figure 1

Only CD47 isoform 2 is detectable on the erythroblast surface during erythropoiesis. (A) The amino acid sequences of the 4 alternatively spliced isoforms of the CD47 C-terminus. (B) HEK293T cells were transfected with CD47 isoform 2, 3 and 4, and CD47 with a deleted C-terminus (Δ Ct) (5 µg DNA/10 cm2 dish). HEK293T cell lysates were separated by SDS-PAGE (106 cells/lane) against the empty vector and untransfected cells. Western blots were probed with anti-CD47out1 (Total), anti-CD47 isoform 2, or anti-CD47 isoform 3 and 4 (n = 3). (C) Erythroblasts were harvested every 24 hours during expansion or (D) during terminal differentiation, lysed and then separated on SDS-PAGE (106 erythroblasts/lane). Western blots were probed with anti-CD47out1 (Total), anti-CD47 isoform 2 or anti-CD47 isoforms 3 and 4. GAPDH was used as a loading control (n = 3). (E) Every 24 hours 5 × 106 erythroblasts were incubated with BRIC126 to immunoprecipitate CD47 expressed on the surface, the eluate was then separated by SDS-PAGE. Western blots were probed with anti-CD47out1 (Total), anti-CD47 isoform 2 or CD47 isoforms 3 and 4 (n = 3). (F) 10 × 106 erythroblasts were taken every 24 hours, lysed and then immunoprecipitated with BRIC126. The eluate was then separated on SDS-PAGE. Western blots were probed with anti-CD47out1 (Total), anti-CD47 isoform 2 or anti-CD47 isoforms 3 and 4 (protein band indicated by an arrow). *Indicates background band. (n = 2).