Figure 4.
Localization of Endogenous SHIP164 on small clusters of vesicular structures. (A) Cartoon of WT (top) and edited SHIP164^mNG (bottom). (B) Western blot (in kD) of control and edited cell clones for SHIP164, mNG, and GAPDH as a loading control. The different motility of SHIP164 in control and edited clones indicates the incorporation of mNG in the edited SHIP164. (C) Cartoon of endogenously edited locus of SHIP164^mNG in HeLa cell. Red, DNA scar as a result of homology-independent targeted insertion; blue, small Gly-Ser linkers; green, mNG sequence (shortened for brevity by forward slashes). (D) Fluorescence image of endogenous SHIP164^mNG (inverted grays) in an edited HeLa knock-in cell. Arrowheads indicate concentration of endogenous SHIP164 at the cell edge. Scale bar, 10 µm (main field) and 2 µm (inset). (E) Single channel (inverted grays) fluorescence images of endogenous SHIP164^mNG and endogenous MPR-mScarlet in a HeLa double-knock-in cell. Scale bar, 10 µm. High-magnification scale bar, 2 µm. (F) Left to right: Fixed fluorescence image of endogenously tagged SHIP164^mNG (magenta) HeLa cell also expressing mito-BFP (green) for alignment in CLEM analysis. Scale bar, 5 µm. Single plane of the area is outlined in yellow. Fluorescence overlay with FIB-SEM image: numbered boxes mark region of interest of eSHIP164^mNG fluorescence to be aligned with EM image. Scale bar of magnification area, 1 µm. Magnification of cluster of vesicles aligned with eSHIP164^mNG fluorescence marked by numbered boxes. Scale bar of EM area, 100 nm. (G) Reconstruction of cluster 1 from panel F above. SHIP164-associated endosome structures, magenta; ER tubules, yellow. (H) Fluorescence image of endogenously tagged SHIP164^mNG in a HeLa knock-in cell also expressing the ER marker RFP-Sec61 (green) demonstrating some SHIP164 foci are in proximity of ER tubules. Scale bar, 2 µm. Source data are available for this figure: SourceData F4.

Localization of Endogenous SHIP164 on small clusters of vesicular structures. (A) Cartoon of WT (top) and edited SHIP164^mNG (bottom). (B) Western blot (in kD) of control and edited cell clones for SHIP164, mNG, and GAPDH as a loading control. The different motility of SHIP164 in control and edited clones indicates the incorporation of mNG in the edited SHIP164. (C) Cartoon of endogenously edited locus of SHIP164^mNG in HeLa cell. Red, DNA scar as a result of homology-independent targeted insertion; blue, small Gly-Ser linkers; green, mNG sequence (shortened for brevity by forward slashes). (D) Fluorescence image of endogenous SHIP164^mNG (inverted grays) in an edited HeLa knock-in cell. Arrowheads indicate concentration of endogenous SHIP164 at the cell edge. Scale bar, 10 µm (main field) and 2 µm (inset). (E) Single channel (inverted grays) fluorescence images of endogenous SHIP164^mNG and endogenous MPR-mScarlet in a HeLa double-knock-in cell. Scale bar, 10 µm. High-magnification scale bar, 2 µm. (F) Left to right: Fixed fluorescence image of endogenously tagged SHIP164^mNG (magenta) HeLa cell also expressing mito-BFP (green) for alignment in CLEM analysis. Scale bar, 5 µm. Single plane of the area is outlined in yellow. Fluorescence overlay with FIB-SEM image: numbered boxes mark region of interest of eSHIP164^mNG fluorescence to be aligned with EM image. Scale bar of magnification area, 1 µm. Magnification of cluster of vesicles aligned with eSHIP164^mNG fluorescence marked by numbered boxes. Scale bar of EM area, 100 nm. (G) Reconstruction of cluster 1 from panel F above. SHIP164-associated endosome structures, magenta; ER tubules, yellow. (H) Fluorescence image of endogenously tagged SHIP164^mNG in a HeLa knock-in cell also expressing the ER marker RFP-Sec61 (green) demonstrating some SHIP164 foci are in proximity of ER tubules. Scale bar, 2 µm. Source data are available for this figure: SourceData F4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal