In all cases, despite detecting a CS-induced decrease in full-size CFTR, we failed to detect Dovitinib cost CFTR fragments post-CS using 12% bis-tris gels (all n=3; data not shown). CS exposure reduces CFTR solubility and induces CFTR relocation to a perinuclear compartment We performed the Western blots shown in Fig. 4 using Nonidet P-40, since this detergent has been found to be superior for extraction of membrane proteins, such as CFTR (28). However, to ensure that we had fully solubilized CFTR after smoke exposure, we also utilized a higher concentration of ionic detergent. Surprisingly, on extraction with a high concentration of SDS (i.e., 10%), the CS-induced reduction in the CFTR signal was no longer observed (Fig. 6A, B), suggesting that CS exposure resulted in CFTR becoming significantly less detergent soluble without inducing CFTR degradation.

The experiments shown in Fig. 6A, B were performed in BHKCFTR cells. To test whether this effect was also observable in airway epithelia, we also exposed polarized CALU3 cells, grown at an air-liquid interface to CS, since these cells endogenously express CFTR and have previously been used for CS-exposure experiments (5, 32). Following CS exposure, a reduction in CFTR levels could be detected in Nonidet P-40- but not SDS-lysed cultures (Fig. 6C). Figure 6. CFTR solubility is altered after CS exposure. A) Western blot showing CFTR from BHKCFTR cells lysed following standard exposure to air or CS using Nonidet P-40, or lysis buffer containing 10% SDS. B) Mean densitometry for CFTR taken from A. n = 6/group. …

To further investigate the change in solubility of CFTR, we compared immunostaining of CFTR after CS exposure with either paraformaldehyde followed by permeabilization with 1% Triton-X or following fixation/permeabilization with methanol using the 596 antibody against CFTR’s NBD2. While CFTR could be detected equally well in air-exposed BHK cultures using either paraformaldehyde or methanol, CFTR could only be detected following methanol fixation post-CS exposure (Fig. 6D). Thus, the pool of CFTR that exhibits reduced solubility post-CS may become accessible to the antibody only following harsher permeabilization with methanol. Chronic smoke exposure has previously been shown to decrease CFTR expression levels in CALU3 cells (5). To investigate whether CFTR remained insoluble to Nonidet P-40 after chronic smoke exposure, we utilized the protocol employed by Cantin et al.

(5) and exposed polarized CALU3 cells to smoke from 1 cigarette every 2 h for 8 h. After this time, CFTR gene expression was significantly reduced (Fig. 6E), and CFTR protein levels declined following lysis in Nonidet P-40 (Fig. 6F, G). However, CFTR protein AV-951 could be recovered after lysis in 10% SDS, suggesting that CFTR remained in a detergent-resistant fraction after 8 h of chronic CS exposure (Fig. 6F, G).