Computational protocol: Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4

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[…] HEK293 cells transiently transfected with HA-CXCR4-YFP were passaged onto poly-l-lysine–coated coverslips and allowed to grow for 24 h. HeLa cells were used to examine the distribution of endogenous CXCR4. Cells were washed once with warm DMEM containing 20 mM HEPES, pH 7.5, and incubated in the same medium for 3–4 h at 37°C. Cells were treated with 30 nM CXCL12 or vehicle for 30 min, fixed with 3.7% paraformaldehyde, and then permeabilized with 0.05% (wt/vol) saponin for 10 min, similar to a protocol we have described previously (). Cells were coincubated with STAM-1, EEA1, or β-arrestin2/3 antibodies. Endogenous CXCR4 in HeLa cells was stained with rat anti-CXCR4 mAb. In brief, after permeabilization and fixation, cells were incubated with 1% BSA in 0.05% saponin-phosphate-buffered saline (PBS) for 30 min at 37°C, followed by incubating with primary antibody for 1 h at 37°C. Primary antibodies for STAM-1 and EEA1 were used at 1:100 dilution and against CXCR4 and β-arrestin2/3 was used at a 1:50 dilution. Cells were washed five times with 0.05% saponin-PBS, followed by incubating with appropriate Alexa-Fluor–conjugated secondary antibodies for 30 min at 37°C. Finally, cells were washed with PBS and fixed again in 3.7% formaldehyde-PBS and mounted onto glass slides using mounting media containing 4,6-diamidino-2-phenylindole. Samples were analyzed using an LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) equipped with a Plan-Apo 63×/1.4 oil lens objective. Images were acquired using a 1.4-megapixel cooled extended spectra range RGB digital camera set at 512 × 512 resolution. Acquired images were analyzed using ImageJ, version 1.41o (National Institutes of Health, Bethesda, MD), and the amount of colocalization between proteins was determined using MetaMorph 7.6 (Molecular Devices, Downingtown, PA). […]

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