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. 2019 Jun 4;10(38):3694–3708. doi: 10.18632/oncotarget.26979

Figure 3. Immunoprecipitation of MAGEA-EVs.

Figure 3

(A) MAGEA4 and MAGEA10 EVs were pulled down from 2K, 16K and 120K fractions using MAGEA4 and MAGEA10-specific antibodies. Immunoprecipitation was performed as depicted in Materials and Methods section and Western blot analysis was performed with anti-E2Tag antibody. Input (10%) and IP are shown. (B) Coomassie Blue staining of SDS-PAGE of immunoprecipitated material of panel (A). The positions of antibody heavy and light chains are shown by arrows. (C) Western blot analysis of EVs purified from COP5 cells ectopically expressing recombinant MAGEA proteins using flotillin, (610820; BD Transduction Laboratories), anti-integrin beta 1 (ab179471; Abcam) and anti-histone H3 (ab1791; Abcam) antibodies. 5% of each EV fraction was loaded to the gel. (D) Coomassie Blue staining of SDS-PAGE of 2K, 16K and 120K fractions isolated from COP5 cells expressing MAGEA4, MAGEA10 and MLVGag proteins, respectively. The position of MLVGag protein in VLPs is shown by arrow. (E) Analysis of immunoprecipitated material from 2K fractions with anti-flotillin and anti-histone H3 antibodies. (F) Analysis of immunoprecipitated material from 120K fractions with anti-TSG101, anti-flotillin, anti-integrin beta 1 and anti-histone H3 antibodies.