Anti egfr antibody

Cells were incubated in DMEM without methionine or cysteine (Met/Cys [-]) for 30–60 min, and in DMEM (Met/Cys [-]) containing a mixture of [³⁵S] methionine and cysteine (Perkin Elmer) for 120 min. Cells were then washed three times in cold DMEM and were incubated in DMEM containing 10% FBS for 0, 4, 8, 12, and 24 h. They were lysed in PBS containing 1% Triton X-100 and protease inhibitor cocktail, and the lysates were incubated with Protein A-conjugated sepharose beads for 1 h and then collected in new tubes. Lysates were incubated with 2 µg of anti-EGFR antibody (Millipore) for 20–24 h and beads were then washed five times in lysis buffer. Bound proteins were then eluted from the beads with 1 × SDS-PAGE sample buffer followed by boiling at 90 °C for 10 min, and were finally analyzed using SDS-PAGE. Dried gels were exposed to a BAS-imaging plate and were scanned using a BAS2500 phosphorimager (Fujifilm). EGFR band intensities were quantified using a software of Multi Gauge Ver2.02 (Fujifilm) or ImageJ. All experiments were repeated three times.

EGFR level in internal vesicles of late endosome was quantified by electron microscopy (EM) as previously described^{32 (link)}. Cells treated with siRNA against PGK1 or not were starved overnight, incubated with anti-EGFR antibody (Millipore, LA22) at 4 °C for 30 min, washed and followed by incubation at 4 °C for additional 30 min with 15 nm protein A-gold (Electron Microscopy Sciences). Cells were then washed, stimulated with 100 ng/ml EGF for 1 h at 37 °C, fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer and processed for EM examination in the EM facility. The ultrasections were viewed using a Jeol JEM-1400 transmission electron microscope.

Immunofluorescence microscopy was performed using standard protocols. Briefly, cells were fixed with 4% PFA in 0.1 M phosphate buffer (pH 7.4) and were then permeabilized in PBS containing 0.1% Triton X-100 and 0.4% BSA or in PBS containing 50 µg/ml digitonin and 0.4% BSA. They were incubated with primary antibodies followed by the appropriate secondary antibodies conjugated with Alexa488, Alexa594, or Alexa647 (Molecular Probes). Fluorescence images were obtained using a confocal microscope (FV1000, Olympus) equipped with a PlanApo N 60× (NA 1.42 oil) and a UPlan FL N 40× (NA 1.3 oil) lenses. To inhibit lysosomal degradation, cells were transfected with siRNAs and treated with E64d and Leupeptin for 6 h and were then fixed for immunofluorescence analyses. Percentages of double positive puncta for EGFR and cathepsin D over cathepsin D positive ones were measured.
For quantification of EGFR signal in GGA2-depleted ARPE-19 cells re-expressing HA-GGA2^kd-resi, fixed cells were immunostained with anti-EGFR antibody (Millipore) without permeabilization, and then immunostained with anti-HA antibody following permeabilization. Four serial x-y images along z-axis, which cover majority of a whole cell, were acquired and projected into a single image. Mean intensity for EGFR signal per cell was quantified using ImageJ software.