Anti p egfr tyr1068

HeLa^Clover-PRP4K cells were plated onto glass coverslips in a 6-well plate and treated overnight with 50 μm chloroquine (Sigma) or stimulated with EGF as above. Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min and immunolabeling was carried out as previously described.^{10 (link)} Fluorescent images were captured with a Zeiss Cell Observer spinning-disk microscope (Intelligent Imaging Innovations, 3i, Boulder, CO, USA) using a 1.4 NA 63 × immersion oil objective lens. Images were processed using only linear adjustments in Adobe Photoshop CS5 and Slidebook (3i) software, which was also used for the analysis of mean fluorescence intensity of immunostaining.
Antibodies used for immunofluorescence were used at the manufacturers recommended dilution unless otherwise noted, and include anti-GFP (Abcam, Toronto, ON, Canada, ab13970) (1:2000 dilution), anti-EEA1 (Cell Signaling, #3288) (1:100 dilution), anti-Rab5 (Cell Signaling, #3547) (1:200 dilution), anti-Rab7 (Cell Signaling, #9367) (1:100 dilution), anti-p62 (Cell Signaling, #7695) (1:400 dilution), anti-LAMP2 (Abcam, ab25631) (1:200 dilution), and anti-p-EGFR (Tyr1068) (Cell Signaling, #3777) (1:800 dilution).