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. 2017 Jun 23;12(6):e0180257. doi: 10.1371/journal.pone.0180257

Fig 5. Effect of co-transfection of a-non-functional OATP1B3 mutant on function and oligomerization.

Fig 5

(A) Membrane fractions isolated from HEK293 cells transiently transfected with OATP1B3-FLAG and OATP1B3-K41C-His and treated with or without DTSSP were solubilized. Immunoprecipitation was performed with Anti-His or Anti-FLAG antibodies and the eluted proteins were detected with Anti-His antibody. (B) HEK293 cells were transiently transfected with 500ng of OATP1B3-K41C-His plus 500ng of pcDNA5/FRT (Mutant), with 500ng of OATP1B3-FLAG plus 500ng of pcDNA5/FRT (WT), or with 500ng of OATP1B3-FLAG plus 500ng of OATP1B3-K41C-His (Co-Transfection). Forty-eight hours later uptake of 10μM E3S or 1μM E17βG was performed at 37°C for 1 min. (C) Surface biotinylated proteins were isolated from HEK293 cells transiently transfected as in (B). Western blot was performed with Anti-His (Mutant-His) or Anti-FLAG (WT-FLAG). Na+/K+ ATPase was used as loading control. (D) Uptake shown in (B) was normalized by the surface expressed mutant-His or WT-FLAG protein determined in (C) after subtracting of empty-vector control uptake.