bioimagesUnknownGlen A. FarrThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-200901021JCB

Cells were infected to express the human Tfn-R and VSV-G-YFP Ts045 (green) and incubated overnight at 40 degrees. Next, cells were BG-Blocked and incubated for 40 minutes at 40 degrees with Tfn-HRP, along with 1uM TMR-STAR to “live label” newly synthesized pump (red). Next, cells were washed and incubated for a further 20 minutes at 40 degrees to remove excess TMR-STAR and to allow for the accumulation of Tfn-HRP into REs. Tfn-HRP loaded REs were ablated on ice by exposing cells to DAB and H2O2 for 1h, while control cells were treated with DAB alone. After the ablation reaction, cells were fixed or incubated in media plus CHX for 1.5h at 31 degrees to allow trafficking of the proteins to the PM. Samples were processed for immunofluorescence with a cocktail of Golgi antibodies (GM130,

SNAP-cells were transfected with a plasmid encoding Ts 045 VSV-G-YFP and incubated for 24h at 40 degrees to accumulate newly synthesized VSV-G in the ER. Next, samples were BG-Blocked, washed and incubated for a short 10 minute recovery period to permit synthesis of new SNAP tagged sodium pump protein. Samples were subjected to 19 degree Golgi block and fixed immediately. Samples were labeled with TMR-STAR (red) and imaged for YFP (green).

Cells were infected to express the human Tfn-R and VSV-G-YFP Ts045 (green) and incubated overnight at 40 degrees. Next, cells were BG-Blocked and incubated for 40 minutes at 40 degrees with Tfn-HRP, along with 1uM TMR-STAR to “live label” newly synthesized pump (red). Next, cells were washed and incubated for a further 20 minutes at 40 degrees to remove excess TMR-STAR and to allow for the accumulation of Tfn-HRP into REs. Tfn-HRP loaded REs were ablated on ice by exposing cells to DAB and H2O2 for 1h, while control cells were treated with DAB alone. After the ablation reaction, cells were fixed or incubated in media plus CHX for 1.5h at 31 degrees to allow trafficking of the proteins to the PM. Samples were processed for immunofluorescence with an antibody against the Tfn-R shown in

bioimagesFigure 5Image 4375 (Figure 5 - D)Image 4378 (Figure 7 - B)Image 4376 (Figure 5 - D)Figure 7Image 4377 (Figure 7 - A)Figure 7 - AFigure 7 - BFigure 5 - DMichael J. CaplanGlen A. FarrIra MellmanMichael HullfalseMembrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells2009-07-20T11:17:27Z2018-11-29T11:17:27Z2018-11-29T11:17:27ZS-JCBD-20090102110.1083/jcb.200901021