Figure S2.
Exogenous SHIP164 forms foci. (A) Live fluorescence (inverted grays) image of a COS-7 cell expressing exogenous 3xFlag-SHIP164^mScarlet. Scale bar, 20 µm. (B) Live image of the cytoplasm of a COS-7 cell expressing exogenous SHIP164^mScarlet (magenta) and the endosomal marker GFP-2xHrsFYVE (green). Arrowheads indicate SHIP164 accumulation juxtaposed to endosomal membrane. Scale bar, 5 µm. (C) Live fluorescence image of a COS-7 cell expressing SHIP164-Halo (magenta) and the dominant negative GFP-Rab5 S34N mutant. Scale bar, 20 µm. (D and E) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of MPR in a cell expressing RFP alone (D) and its abnormal localization in a cell expressing both RFP and SHIP164 (E). Cells were immunolabeled with antibodies against MPR (green) and GM130 (magenta). Both the merge image and the single channels are shown. Arrowheads indicate ectopic accumulations of MPR in cells over-expressing SHIP164. Scale bar, 5 µm. (F and G) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of sortilin in a cell expressing RFP alone (F) and its abnormal localization in a cell expressing both RFP and SHIP164 (G). Cells were immunolabeled with antibodies against sortilin (green) and GM130 (magenta). Both the merge image and the single channels are shown. Arrowheads indicate ectopic accumulations of sortilin in cells over-expressing SHIP164. Scale bar, 5 µm. (H and I) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of TGN46 in a cell expressing RFP alone (H) and its unaffected localization in a cell expressing both RFP and SHIP164 (I). Cells were immunolabeled with antibodies against TGN46 (green) and GM130 (magenta). Both the merge image and the single channels are shown. Scale bar, 5 µm. (J) Merge fluorescence image of endogenous SHIP164^mNG (green) and exogenous SHIP164^mScarlet (magenta) in an edited HeLa knock-in cell to demonstrate overlap of the two fluorescence signals (arrowheads). Scale bar, 10 µm. High magnification of the indicated region is shown at right where the individual channels are shown as inverted grays. Scale bar, 2 µm. (K) Merge fluorescence image of endogenous MPR-mScarlet and endogenous SHIP164^mNG in a HeLa double-knock-in cell demonstrating partial colocalization of the two fusion proteins at the cell edge but not with pulsed EGF-647 (blue) foci. Scale bar, 10 µm. High magnification of the indicated region is shown at right and the individual channels are shown as inverted grays. Open arrowheads indicate overlapping fluorescence, and blue arrowheads an EGF-647–positive endocytic structure. Scale bar, 2 µm.

Exogenous SHIP164 forms foci. (A) Live fluorescence (inverted grays) image of a COS-7 cell expressing exogenous 3xFlag-SHIP164^mScarlet. Scale bar, 20 µm. (B) Live image of the cytoplasm of a COS-7 cell expressing exogenous SHIP164^mScarlet (magenta) and the endosomal marker GFP-2xHrsFYVE (green). Arrowheads indicate SHIP164 accumulation juxtaposed to endosomal membrane. Scale bar, 5 µm. (C) Live fluorescence image of a COS-7 cell expressing SHIP164-Halo (magenta) and the dominant negative GFP-Rab5 S34N mutant. Scale bar, 20 µm. (D and E) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of MPR in a cell expressing RFP alone (D) and its abnormal localization in a cell expressing both RFP and SHIP164 (E). Cells were immunolabeled with antibodies against MPR (green) and GM130 (magenta). Both the merge image and the single channels are shown. Arrowheads indicate ectopic accumulations of MPR in cells over-expressing SHIP164. Scale bar, 5 µm. (F and G) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of sortilin in a cell expressing RFP alone (F) and its abnormal localization in a cell expressing both RFP and SHIP164 (G). Cells were immunolabeled with antibodies against sortilin (green) and GM130 (magenta). Both the merge image and the single channels are shown. Arrowheads indicate ectopic accumulations of sortilin in cells over-expressing SHIP164. Scale bar, 5 µm. (H and I) Fluorescence images of RPE-1 cells demonstrating normal Golgi complex localization of TGN46 in a cell expressing RFP alone (H) and its unaffected localization in a cell expressing both RFP and SHIP164 (I). Cells were immunolabeled with antibodies against TGN46 (green) and GM130 (magenta). Both the merge image and the single channels are shown. Scale bar, 5 µm. (J) Merge fluorescence image of endogenous SHIP164^mNG (green) and exogenous SHIP164^mScarlet (magenta) in an edited HeLa knock-in cell to demonstrate overlap of the two fluorescence signals (arrowheads). Scale bar, 10 µm. High magnification of the indicated region is shown at right where the individual channels are shown as inverted grays. Scale bar, 2 µm. (K) Merge fluorescence image of endogenous MPR-mScarlet and endogenous SHIP164^mNG in a HeLa double-knock-in cell demonstrating partial colocalization of the two fusion proteins at the cell edge but not with pulsed EGF-647 (blue) foci. Scale bar, 10 µm. High magnification of the indicated region is shown at right and the individual channels are shown as inverted grays. Open arrowheads indicate overlapping fluorescence, and blue arrowheads an EGF-647–positive endocytic structure. Scale bar, 2 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal