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. 2019 Jul 29;19:267–280. doi: 10.1016/j.isci.2019.07.039

Figure 2.

Figure 2

TMEM88 Is Present in the Golgi, Plasma Membrane, and Digitonin-Resistant EEA1+ MVBs in Cardiac Progenitor Cells

(A) Immunofluorescence of TMEM88 and NKX2.5 in H1 embryonic stem cell (ESC)-derived day 7 cardiomyocyte progenitor cells. NKX2.5 is restricted to the nucleus, shown by Hoechst 33342 staining in the right panel. White arrow and arrowhead show localization of TMEM88 to the plasma membrane and MVB, respectively.

(B) Immunoelectron microscopy demonstrating presence of endogenous TMEM88 in MVB (arrows) in H1 ESC-derived day 7 cardiomyocyte progenitor cells. Arrows indicate gold-conjugated anti-TMEM88 antibody staining.

(C) Immunofluorescence of TMEM88 and an endosome marker, EEA1, in H1 ESC-derived day 7 differentiating cardiac progenitors. Cells were permeabilized with RB buffer supplemented with digitonin (25 μg/mL), fixed, and analyzed by immunofluorescence with the indicated antibody (Vinyoles et al., 2014).

(D) Western blots of digitonin-soluble fraction (DSF) and digitonin-resistant fraction (DRF) of day 5 ESC-derived differentiating cardiac progenitor cells using the CHIR99021/IWP2 protocol described in Methods. Membrane marker Na/K ATPase was used as a loading control for the DRF.