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. 2010 Aug 23;190(4):565–574. doi: 10.1083/jcb.201004060

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© 2010 Lauffer et al.

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Figure 4. — Transgenic rescue of β₂AR recycling by recombinant SNX27. (A) Immunoblot showing depletion of endogenous SNX27 by silencing relative to negative control siRNA (lanes 1 and 2), and replacement by cotransfection of SNX27-GFP but not GFP control plasmid (lanes 3 and 4). Electrophoretic mobilities of endogenous and SNX27 and recombinant SNX27-GFP are indicated by arrows. K, kilodaltons. (B) Flow cytometric analysis showing rescue of FLAG-β₂AR recycling in SNX27 knockdown cells by cotransfection of either isoform of recombinant rat SNX27, or their GFP-tagged counterparts. (C and D) Verification of transgenic rescue using dual-channel fluorescence flow cytometry and gating of recycling data based on recombinant SNX27 expression. (C) A representative fluorescence intensity histogram of the GFP channel. The region indicated by (+) represents the populations used to verify transgenic rescue of recycling. (D) FLAG-β2AR recycling measured specifically in the SNX27/GFP (+) population. Error bars indicate SEM. P-values: Student’s t test with GFP control; n = 4–6 experiments.