Abstract
CFTR biogenesis starts with the co-translational folding and insertion into the endoplasmic reticulum (ER) membrane, followed by core-glycosylation and exit to the Golgi. CFTR glycan moieties are then processed along the Golgi, generating the mature membrane-resident form. This process is known as processing or maturation and a simple Western blot (WB) analysis distinguishes the core-glycosylated immature ER-specific form of CFTR (band B) from its fully-glycosylated mature post-ER form (band C). This biochemical assay is thus used to monitor CFTR exit from the ER. Moreover, as it provides quantitative information on steady-state levels, it can be used to assess the impact on CFTR of chemicals or molecules (over or underexpression of a given gene). Our goal was to optimize WB to establish a robust, simple and sensitive method to analyse CFTR expressed in cell lines (epithelial and non-epithelial) grown on 96-well plates which can be used as a secondary test after high-content siRNA screen for proteins involved in CFTR trafficking. Different cell lines were tested here: Calu-3, CFBE and BHK stably expressing either wt- or F508del-CFTR. WB was performed as previously but using benzonase to shear DNA. In each lane of a 7% (w/v) SDS-PAGE gel, contents of 1 to 6 wells from a 96-well plate were applied (about 7.5 mg to 45 mg protein). After electrophoresis and electroblotting, the membrane was blocked and incubated with the 596 anti-CFTR antibody (CFF) at 1:1000, followed by incubation with secondary antibody. Detection was done using West Pico System (Pierce). First, we determined the minimal amount of cells needed to detect CFTR by WB and we found that ~4 x 104 cells (number present in one well) is enough to detect both bands B and C of CFTR in the above cells lines. We then determined the sensitivity of this assay to effects caused by siRNA transfection by using as control siRNAs against CFTR (Silencer Select, Ambion, ref. s2945, s2947). We found a significant decrease (~65 and ~85% respectively) in the amounts of band B and C for wt-CFTR, after 48h of transfection with 1.2 pmol of siRNA. We conclude that this large-scale WB assay is robust to test the effect of different siRNAs against genes possibly affecting the trafficking of wt- and F508del-CFTR.