Cells were washed twice in Staining Buffer and were analyzed using LSR 1 or LSRII (BD Bioscience) in the Stanford Shared FACS Facility.
Proteins were eluted in Laemmli sample buffer and were analyzed by SDS-PAGE followed by immunoblot with indicated antibodies.
The lysates were prepared from dissected sensory epithelia using hypotonic buffer and were analyzed by Western blotting either before or after endoglycosidaseF treatment (Roche, Indianapolis, IN).
Samples were washed in BSA buffer and were analyzed on a FACS Calibur.
After washing cells were resuspended in 400 μl of FACS buffer and were analyzed using a LSRII FACS machine (BD Bioscience, Heidelberg, Germany).
Following rigorous washing steps, samples were resuspended in 0.5 ml of FACS buffer and were analyzed using a LSRII FACS machine (BD Bioscience, Heidelberg, Germany).
Proteins were then purified using HisSpinTrap™ columns (GE Healthcare) using 25 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole, pH 7.4, as equilibration and wash buffer and 25 mM Tris-HCl, 500 mM NaCl, and 500 mM imidazole, pH 7.4, as elution buffer and were analyzed by SDS-PAGE using precast NuPAGE® gels (Invitrogen) and Western blotting.
After fixation in 1% paraformaldehyde for 10 min at 25°C, cells were washed in PBS and permeabilized for 10 min at room temperature in saponin buffer (0.5% (weight/volume) saponin, 5% FBS and 10 mM HEPES, pH 7.4, in PBS) Permeabilized cells were incubated for 45 min with phycoerythrin-conjugated Ab to TCR in saponin buffer, were washed in saponin buffer and were analyzed on a FACS Calibur.
The cells were washed in PhosFlow Perm/Wash Buffer I, and were analyzed by flow cytometry, as already described.
