Fig. 7: Interaction of RUFY3 and RUFY4 with dynein-dynactin.
From: RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin
a HEK293T cells were transfected with plasmids encoding GFP (negative control), GFP-BICD2 (positive control), RUFY3-GFP or RUFY4-GFP. Cell extracts were analyzed by immunoprecipitation (IP) with antibody to GFP, followed by immunoblotting (IB) for endogenous dynein intermediate chain (DIC) and endogenous p150Glued subunit of dynactin. Ponceau S staining shows the levels of immunoprecipitated GFP-tagged proteins. The experiment shown in this panel is one of two with similar results. b Extracts of HEK293T cells were incubated with recombinant 6His-StrepII-sfGFP (abbreviated 6His-GFP in the figure) (negative control), His6-StrepII-sfGFP-BICD225-400 (His6-GFP-BICD225-400) (positive control) or 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3), pulled down (PD) with Strep-Tactin agarose, and immunoblotted for endogenous dynein intermediate chain (DIC), the endogenous p150Glued of dynactin, or GFP. The GFP used to make these constructs is a variant named sfGFP, for super-folder GFP. The experiment shown in this panel is one of two with similar results. c Glutathione-Sepharose preloaded with purified, recombinant GST (negative control), GST-DLIC1 or GST-DLIC1-CT (C-terminal domain) was incubated with purified, recombinant 6His-StrepII-sfGFP-RUFY3 (6His-GFP-RUFY3). Bound proteins were detected by immunoblotting with antibodies to GFP and GST. The positions of molecular mass markers (in kDa) in panels a-c are indicated at left. d Live-cell images of HeLa cells co-expressing RUFY3-mCherry or RUFY4-mCherry (magenta) without or with GFP-p150Glued-CC1 (green). Single-channel images are shown in grayscale. Scale bars: 10 μm. Arrows point to RUFY proteins at cell tips. This experiment is one of two with similar results.
