Figure 6.
Deoxynojirimycin-enhances release of YFP-PrP* for ER-export by a pathway that is dependent on Tmp21 and results in transient exposure on the cell surface. (A) Images of representative YFP-PrP* CER-GalT NRK cells that were subjected to Tmp21 siRNA knockdown treatment followed by treatment with 2.5 mM deoxynojirimycin (“+DNJ”) for 45 min or 0.1 µM thapsigargin (“+TG”) for 30 min before fixation and immunofluorescence against Tmp21. Each panel shows two adjacent cells in the same field of view where one cell was fully depleted for Tmp21 (cell on right) and the other cell was not (cell on left). For cells treated with DNJ for 45 min, quantification of 107 randomly imaged Tmp21-depleted cells revealed that YFP-PrP* is localized to the ER in 100% of the Tmp21-depleted cells. For cells treated with TG for 30 min, quantification of 53 randomly imaged Tmp21-depleted cells revealed that YFP-PrP* is localized to the ER in 100% of the Tmp21-depleted cells, confirming a finding previously described (Satpute-Krishnan et al., 2014). (B) Time-lapse imaging of an NRK cell stably expressing both YFP-PrP* and lysosome-marker Lamp1-mCherry, labeled “YFP-PrP* NRK,” or Lamp1-mCherry alone, labeled “Parent NRK.” Cells were incubated with 250 nM bafilomycin A1 and AlexaFluor (TM) 647-conjugated anti-GFP antibody (αGFP-AF647). (C) Time-lapse imaging of an NRK cell stably expressing both YFP-PrP* and Lamp1-mCherry, incubated with 2.5 mM 1-deoxynojirimycin, 250 nM bafilomycin A1, and αGFP-AF647. Identical imaging parameters were applied for all cells imaged for B and C. Scale bar, 10 μm.

Deoxynojirimycin-enhances release of YFP-PrP* for ER-export by a pathway that is dependent on Tmp21 and results in transient exposure on the cell surface. (A) Images of representative YFP-PrP* CER-GalT NRK cells that were subjected to Tmp21 siRNA knockdown treatment followed by treatment with 2.5 mM deoxynojirimycin (“+DNJ”) for 45 min or 0.1 µM thapsigargin (“+TG”) for 30 min before fixation and immunofluorescence against Tmp21. Each panel shows two adjacent cells in the same field of view where one cell was fully depleted for Tmp21 (cell on right) and the other cell was not (cell on left). For cells treated with DNJ for 45 min, quantification of 107 randomly imaged Tmp21-depleted cells revealed that YFP-PrP* is localized to the ER in 100% of the Tmp21-depleted cells. For cells treated with TG for 30 min, quantification of 53 randomly imaged Tmp21-depleted cells revealed that YFP-PrP* is localized to the ER in 100% of the Tmp21-depleted cells, confirming a finding previously described (Satpute-Krishnan et al., 2014). (B) Time-lapse imaging of an NRK cell stably expressing both YFP-PrP* and lysosome-marker Lamp1-mCherry, labeled “YFP-PrP* NRK,” or Lamp1-mCherry alone, labeled “Parent NRK.” Cells were incubated with 250 nM bafilomycin A1 and AlexaFluor (TM) 647-conjugated anti-GFP antibody (αGFP-AF647). (C) Time-lapse imaging of an NRK cell stably expressing both YFP-PrP* and Lamp1-mCherry, incubated with 2.5 mM 1-deoxynojirimycin, 250 nM bafilomycin A1, and αGFP-AF647. Identical imaging parameters were applied for all cells imaged for B and C. Scale bar, 10 μm.

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