Ha 11 antibody

Cells were seeded in 96-well plates. VX-809 (#S1565, Selleckchem, Houston, TX, USA, 2.5 µM) or dimethylsulfoxide (DMSO) was added 24 h later for selected experiments. For the dose-response of VX-809, cells were treated with a concentration range of 50 nM to 50 µM with constant DMSO. 24 h later, cells were dissociated to single cells with 0.25% Trypsin-EDTA for 2 min at 37 °C and transferred to a conical 96-well plate. PM and total staining were performed in parallel. For PM staining, cells were stained with HA.11 antibody (#901515, Biolegend 1:1000) at 4 °C, then fixed with IC fixation buffer (#00-8222-49, eBioscience, Waltham, MA, USA) and followed by Alexa-488 secondary antibody (#A-11001, Thermo Fischer Scientific, 1:500). For total staining cells were fixed and permeabilized prior to primary antibody. Per well 10.000 events were measured with the Guava easyCyte HT and analyzed in Guava InCyte. HEK293T cells, not overexpressing any CFTR variant were used as a negative control to determine the positive fraction. Plasma membrane density was determined as the percentage of PM CFTR positive cells (%gated) * Median Fluorescent Intensity of the PM CFTR (MFI), relative to WT CFTR. Traffic efficiency (TE) by VX-809 was compared to DMSO (ΔTE = TE_VX-809 − TE_DMSO), with TE = % PM/ % Total CFTR (% of WT DMSO).

For PM staining, cells were blocked with 1% BSA-PBS and incubated with HA.11 antibody (#901515, Biolegend, San Diego, CA, USA, 1:1000) at 4 °C on living cells. Next, cells were fixed (4% PFA) followed by Alexa-488 secondary antibody (#A-11001, Thermo Fischer Scientific, 1:500). Next, cells were fixed again, followed by permeabilization and HA.11 primary antibody (1:1000). Total anti-HA stained CFTR was visualized using Alexa-633 secondary antibody (#A-21050, Thermo Fischer Scientific, 1:500). Nuclei were stained with DAPI (4′,6-diamidino-2-fenylindole, #D1306, Thermo Fischer Scientific, 1:2000) and sections analyzed by confocal microscopy.