Figure 3.

The TMH4,10-14A mutant is expressed in HeLa cells with overall conformation very similar to WT P-gp. (A) Cartoon representation of TMH4 (green) and TMH10 (cyan) with Taxol bound in the center of the binding pocket (pdb.6QEX). Residues shown as stick models were mutated to Ala to generate the TMH4,10-14A mutant. (B) HeLa-S3 cells were transduced with WT, TMH4,10-14A mutant BacMam baculovirus, stained with the human P-gp-specific monoclonal antibody MRK-16, and analyzed using flow cytometry as described in the Materials and Methods section. Cells expressing WT and TMH4,10-14A mutant P-gp stained with MRK-16 monoclonal antibody are indicated with black and red curves. (C) Expression of WT P-gp is taken as 100%, and the relative expression of TMH4,10-14A mutant P-gp was calculated. Three to five independent replicates were quantified, and error bars show SD. (D) The overall conformation of the TMH4,10-14A mutant is the same as WT protein. The conformation-sensitive monoclonal antibody UIC2 reactivity assay was performed in the presence of DMSO (solvent), a substrate cyclosporine A (10 µM), or an inhibitor tariquidar (10 µM) as described previously [3,4,5,6,7]. (E) Western blot of lysates of HeLa cells transduced with BacMam baculovirus expressing WT and TMH4,10-14A mutant P-gp using the C219 antibody. The lysate of 60,000 cells expressing untransduced cells (lane 1), WT (lane 2), and TMH4,10-14A mutant P-gp (lane 3) were loaded. GAPDH expression was used as a loading control. The PageRuler prestained protein ladder from Thermo Fisher Scientific (Waltham, MA, USA) was used. All these experiments were performed in triplicate. (F) Bar graph representing the quantification of the P-gp levels (in panel D) with error bar showing the SD of three independent experiments. The uncropped blots and the molecular weight marker for Figure 3E are shown in Figures S4 and S5.