2.4. CFTR Immunoprecipitation

Full-length CFTR was immunoprecipitated from whole-cell lysates from CFBE41o− cells stably expressing wt-, F508del-, F508del-4RK-CFTR, or DD/AA-CFTR. Parental CFBE41o− cells were used to define the background using the anti-CFTR monoclonal antibody 596 coupled with rProtein G agarose beads. For the coupling antibody-beads, the anti-CFTR 596 was incubated with beads at a final concentration of 1 µg/mL at RT for 1 h with rocking. Beads were washed with 10 volumes of sodium borate (0.1 M) pH 9 and resuspended again in 10 volumes of sodium borate (0.1 M) pH 9 with dimethyl pimelimidate 2 HCl (DMP) (Thermo Scientific, #21667, Waltham, MA, USA) at the final concentration of 20 mM. The DMP and antibody bound-beads were mixed for 30 min at RT. The reaction was terminated by washing the beads once in ethanolamine (0.2 M) pH 8 and twice in PBS. Finally, beads were resuspended in PBS with 0.02% (w/v) sodium azide and stored at 4 °C.

For in vivo cross-linking, immunoprecipitations were performed in the presence of cells incubated with the cleavable chemical cross-linker dithiobis(succinimidyl propionate) (DSP) (Thermo Scientific, #22585) prior to lysis.

To isolate proteins interacting with CFTR AFT sequence motifs, 10-aminoacid long synthetic peptides conjugated with agarose beads (ProteoChem™, g4101, Hurricane, UT, USA), and containing the CFTR sequence around each of the AFTs, either the wild-type sequence (non-mutated AFTs: R29–CKGYRQRLEL, R516–CDEYRYRSVI, R555–CGGQRARISL and R766–CLQARRRQSV) or the Arg-to-Lys substituted versions (mutated AFTs: K29–CKGYKQRLEL, K516–CDEYKYRSV, K555–CGGQRAKISL, and K766–CLQARRKQSV) were custom-synthesized by CASLO ApS (Kongens Lyngby, Denmark). The pull-down was performed using either a mixture of the non-mutated peptide-conjugated beads or of the mutated peptide-conjugated beads in lysates from CFBE parental cells.

For the preparation of cell lysate for immunoprecipitation or peptide pull-down, cells were washed in cold PBS supplemented with 0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 7.2, incubated for 30 min at 4 °C in either Triton lysis buffer (TBS) (25 mM Tris-HCl pH 7.4, 150 mM NaCl₂, 1% (v/v) Triton-X 100, supplemented with protease inhibitors) in the case of full-length CFTR immunoprecipitation or PD-buffer (Tris-HCl 50 mM pH 7.4; NaCl₂ 100 mM; glycerol 10% (v/v); NP40 1% (v/v) supplemented with protease inhibitors) for peptide pull-down, scraped, and pelleted. The supernatant from the cell lysate was pre-cleared for 1 h at 4 °C with agarose beads, followed by incubation with anti-CFTR 596 antibody cross-linked to rProtein G agarose beads or peptide-conjugated beads overnight at 4 °C. Finally, the sample was washed twice with wash buffer (Tris-HCl 100 mM; NaCl₂ 300 mM) supplemented with 1% (v/v) Triton-X100 and twice with wash buffer without Triton. Proteins were eluted in DTT (50 mM) for 15 min at RT with rocking, followed by incubation for 5 min at 37 °C with Tris-HCl 90 mM pH 7.2, or in the case of peptide pull-down, proteins were eluted in glycine (50 Mm) for 15 min at RT followed by incubation with Tris-HCl (90 mM pH 7.2) 5 min at 37 °C.