Fig 3. CXCR4-[P2G]CXCL12 intermolecular cross-linking efficiencies detected by nonreducing SDS-PAGE and Western blotting of pulled down complexes.

(A) Nonreducing SDS-PAGE and Western blot of His-pull-downs of select combinations of CXCR4 and [P2G]CXCL12 cysteine mutants. The Flag-CXCR4-T4L receptor and [P2G]CXCL12-HA chemokine were detected by LI-COR IRDye conjugated secondary antibodies on a single blot (emission wavelength of 680 nm and 800 nm, visualized in red and green, respectively). Emitted fluorescence detected at 800 nm and 680 nm from the same band of the Western blot is indicative of the receptor and chemokine co-migrating on the gel and thus cross-linked. Images are representative of n = 3 independent replicates. (B) Comparison of cross-linking efficiency determined by flow cytometry (black bars) and Western blotting (green bars). Crosslinking efficiency by Western blotting is given by the anti-HA:IR800 fluorescence intensity. (C) The percentage of cross-linked receptor obtained from Western blotting of the pull-down samples, calculated as the ratio of the IR680 (red) signal intensity of the upper Flag band to the total receptor IR680 signal. Data in (B) and (C) are the mean and SEM of n = 3 independent replicates. The underlying numerical data for each figure panel can be found in S1 Data.