AlexaFluor-555-conjugated CTxB was internalized for 10 min at 37��C, cells were washed, and chased for 60 min. D) Vps52 knockdown redistributes CI-MPR to puncta that also contain SHIP164. CI-MPR was detected with a monoclonal antibody and Stx6 with a rabbit polyclonal antiserum. Bar, 10 ?m. Figure S5: Trafficking defects in cells co-overexpressing Stx6 and SHIP164. A) EGF trafficking is unaffected by Stx6-SHIP164 co-overexpression. HEK293 cells were cotransfected with myc-Stx6 and GFP-SHIP164 for 24 h, incubated with AlexaFluor-555-conjugated EGF for 2 min, washed with PBS and chased in full Ergoloid
medium at 37��C for the indicated times. Asterisks indicate control cells that are not transfected or that express only GFP-SHIP164.
The transferrin receptor (TfR) is trapped in Stx6-SHIP164 structures. HEK293 cells were cotransfected with GFP-SHIP164 and myc-Stx6 for 24 h and processed for confocal immunofluorescence microscopy. TfR was detected with a monoclonal anti-TfR antibody. C) Internalized anti-TGN46 antibody colocalizes with Stx6-SHIP164 structures. HEK293 cells were transfected with TGN46, myc-Stx6 and GFP-SHIP164 for 24 h. Cells were incubated with 1 ?g/mL sheep www.selleckchem.com
polyclonal anti-TGN46 antiserum for 10 min, washed once with PBS, and fixed and processed for confocal immunofluorescence microscopy. Bar, 10 ?m. Video S1: Three-dimensional tomography of a tubulated endosome. HEK293 cells cotransfected with GFP-SHIP164 and myc-Stx6 were processed for CLEM. Individual frames from this video are shown in Figure 10A (panel 2). Please note: Wiley-Blackwell are not responsible for the content Compound C ic50
or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. ""This experiment was conducted to determine whether increasing the net energy (NEL) of a total mixed ration (TMR) with mainly unsaturated fat from corn distillers dried grains with solubles (DDGS) vs. rumen inert (RI)-saturated fat has similar impacts on animal performance. The experiment was an incomplete Youden square with three treatments and four 28-days periods, completed on a large commercial dairy using three early lactation pens each with approximately 380 multiparity cows. The TMR for all treatments was the same, except for 150?g/kg dry matter (DM) of each TMR which contained 90?g/kg high-protein DDGS (HPDDGS) and 60?g/kg beet pulp (i.e. low-fat control diet; LFC); 150?g/kg DDGS (i.e. high-fat diet with unsaturated fat; HFU); or 111?g/kg HPDDGS, 20?g/kg beet pulp and 19?g/kg RI fat (i.e. high-fat diet with saturated fat; HFS). The DM intake was highest (p?<?0.05) for HFU-fed cows. Milk, fat and true protein yields, as well as milk energy output, were higher (p?<?0.01) when cows were fed HFS vs. HFU and LFC diets. Milk true protein concentration was lowest (p?<?0.</div>