Lysosomal pH was measured using LysoSensor Yellow/Blue dextran
(Molecular Probes) per recommended protocol. HEK293 cells were treated for
~4.5 h with V, RB, P1, or RBP1. Cells were washed with
PBS, trypsinized, centrifuged and resuspended in DMEM. Cells were counted
using Trypan Blue and the Invitrogen Countess Cell Counting chamber system
and adjusted to 1 × 10^6 cells/ml. 1 mL cell aliquots were pelleted
by centrifugation, and pellets were resuspended in ~50 μl residual
DMEM. 50 μl of 2 mg/ml Yellow/ Blue dextran in DMEM was added to each
cell pellet for a final concentration of 1 mg/ml dextran, excluding a
no-dextran control, which received DMEM only. Cells were incubated for 2 h
at 37°C, 5% CO2, washed × 3 with Hank’s buffered salt
solution (HBSS), and resuspended in 1 mL HBSS. Samples (100/well) were
loaded on a black 96-well plate (8 wells per each) and fluorescence was
monitored using a Clariostar microplate reader with emission wavelengths at
452 nm and 521 nm, and excitation at 335 nm. The ratio of emissions at
452/521 nm was calculated for each sample, and pH values were calculated
from a linear calibration curve. The calibration curve was generated by
incubating cells for 10 min at 37°C, prior to dextran loading, with
10 uM monensin and 10 uM nigericin in MES buffers (5 mM NaCl, 115 mM KCL,
1.3 mM MgSO4, 25 mM MES), with pHs ranging from 3.5 – 7.0 (Diwu et al., 1999 (link)). r2 =
0.913
(Molecular Probes) per recommended protocol. HEK293 cells were treated for
~4.5 h with V, RB, P1, or RBP1. Cells were washed with
PBS, trypsinized, centrifuged and resuspended in DMEM. Cells were counted
using Trypan Blue and the Invitrogen Countess Cell Counting chamber system
and adjusted to 1 × 10^6 cells/ml. 1 mL cell aliquots were pelleted
by centrifugation, and pellets were resuspended in ~50 μl residual
DMEM. 50 μl of 2 mg/ml Yellow/ Blue dextran in DMEM was added to each
cell pellet for a final concentration of 1 mg/ml dextran, excluding a
no-dextran control, which received DMEM only. Cells were incubated for 2 h
at 37°C, 5% CO2, washed × 3 with Hank’s buffered salt
solution (HBSS), and resuspended in 1 mL HBSS. Samples (100/well) were
loaded on a black 96-well plate (8 wells per each) and fluorescence was
monitored using a Clariostar microplate reader with emission wavelengths at
452 nm and 521 nm, and excitation at 335 nm. The ratio of emissions at
452/521 nm was calculated for each sample, and pH values were calculated
from a linear calibration curve. The calibration curve was generated by
incubating cells for 10 min at 37°C, prior to dextran loading, with
10 uM monensin and 10 uM nigericin in MES buffers (5 mM NaCl, 115 mM KCL,
1.3 mM MgSO4, 25 mM MES), with pHs ranging from 3.5 – 7.0 (Diwu et al., 1999 (link)). r2 =
0.913