Figure S4.
Manipulation of luminal pH of the TGN and its effect on p75-GFP processing.(A) Schematic structure of a luminal pH sensor for the TGN. (B) Calibration curve of B4GALT1-se-pHluorin-tagRFPt pH sensor. Sections of Tg(cldn15la:qf2)pd1142;Tg(quas:B4GALT1-pHluorin-tagRFPt)pd1212 larvae were permeabilized overnight in PBS-0.5% Triton-X100 and then for 20 min in PBS-1% Triton-X100. Afterward, sections were equilibrated in pH-calibrated phosphate-citrate buffer in 20% glycerol for ≥4 h and imaged by confocal microscopy. Image acquisition parameters were set at pH 7.5 to allow high dynamic range with no saturation for each channel. All subsequent imaging for standard curve analysis and live in vivo imaging were then conducted with identical hardware and software parameters. For quantitation of ratiometric imaging of the sensor, raw 16-bit confocal data were used. Curve fitting and pH interpolation was conducted using a sigmoidal model. R2 = 0.8806. n ≥ 5 animals representing ≥25 enterocytes. Error bars are 1 SEM. (C) Representative images from standard curve analysis from B. Scale bars are 10 µm. (D and E)Tg(cldn15la:qf2)pd1142;Tg(quas:B4GALT1-pHluorin-tagRFPt)pd1212 larvae were gavaged with 1% DMSO, 25 µM monensin, 1 µM bafilomycin, or 20 µM nigericin and then allowed to recover for 1 h. Larvae were then mounted in agarose and allowed to recover from tricaine anesthesia for ≥30 min. Media were then replaced with egg water containing the same concentration of inhibitor as gavage conditions. Larvae were then live imaged with no anesthesia within 1 h later. (D) Representative live images. (E) Quantitation of TGN pH values from larvae treated with DMSO, monensin (Mon), bafilomycin (Baf), or nigericin (Nig). ***, P < 0.001; *, P < 0.05; one-way ANOVA. n ≥ 6 animals, representing ≥30 cells. Data points are average pH values for individual animals from randomly selected TGN ROIs. Scale bars are 10 µm. (F and G) Larvae expressing p75-GFP were subjected to a 1-h TGN release in the presence of 0.75% DMSO, 750 nM bafilomycin (Baf), 25 µM monensin (Mon), or 100 mM NH4+. (G) After the 1-h treatment, p75-GFP was immunoprecipitated and analyzed by Western blot. The upper band represents the mature processed form of p75-GFP that is present at steady-state (24 h after induction). The lower bands represent immature p75-GFP intermediates.

Manipulation of luminal pH of the TGN and its effect on p75-GFP processing. (A) Schematic structure of a luminal pH sensor for the TGN. (B) Calibration curve of B4GALT1-se-pHluorin-tagRFPt pH sensor. Sections of Tg(cldn15la:qf2)pd1142;Tg(quas:B4GALT1-pHluorin-tagRFPt)pd1212 larvae were permeabilized overnight in PBS-0.5% Triton-X100 and then for 20 min in PBS-1% Triton-X100. Afterward, sections were equilibrated in pH-calibrated phosphate-citrate buffer in 20% glycerol for ≥4 h and imaged by confocal microscopy. Image acquisition parameters were set at pH 7.5 to allow high dynamic range with no saturation for each channel. All subsequent imaging for standard curve analysis and live in vivo imaging were then conducted with identical hardware and software parameters. For quantitation of ratiometric imaging of the sensor, raw 16-bit confocal data were used. Curve fitting and pH interpolation was conducted using a sigmoidal model. R2 = 0.8806. n ≥ 5 animals representing ≥25 enterocytes. Error bars are 1 SEM. (C) Representative images from standard curve analysis from B. Scale bars are 10 µm. (D and E)Tg(cldn15la:qf2)pd1142;Tg(quas:B4GALT1-pHluorin-tagRFPt)pd1212 larvae were gavaged with 1% DMSO, 25 µM monensin, 1 µM bafilomycin, or 20 µM nigericin and then allowed to recover for 1 h. Larvae were then mounted in agarose and allowed to recover from tricaine anesthesia for ≥30 min. Media were then replaced with egg water containing the same concentration of inhibitor as gavage conditions. Larvae were then live imaged with no anesthesia within 1 h later. (D) Representative live images. (E) Quantitation of TGN pH values from larvae treated with DMSO, monensin (Mon), bafilomycin (Baf), or nigericin (Nig). ***, P < 0.001; *, P < 0.05; one-way ANOVA. n ≥ 6 animals, representing ≥30 cells. Data points are average pH values for individual animals from randomly selected TGN ROIs. Scale bars are 10 µm. (F and G) Larvae expressing p75-GFP were subjected to a 1-h TGN release in the presence of 0.75% DMSO, 750 nM bafilomycin (Baf), 25 µM monensin (Mon), or 100 mM NH4+. (G) After the 1-h treatment, p75-GFP was immunoprecipitated and analyzed by Western blot. The upper band represents the mature processed form of p75-GFP that is present at steady-state (24 h after induction). The lower bands represent immature p75-GFP intermediates.

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