Anti tubulin

The cells were treated with varying concentrations of phosvitin, PPP or 0.5 mg/mL lactoferrin for 72 h. At the end of the incubation period, the culture medium was removed and the cells lysed in boiling hot Laemmle’s buffer containing 50 μM dithiothreitol (DTT, a reducing agent) and 0.2% Triton-X-100 to prepare samples for western blot, as described before [25 (link)]. These cell lysates were run in SDS-PAGE, blotted to nitrocellulose membranes and immunoblotted with antibodies against alkaline phosphatase (ALP; mouse monoclonal antibody from Santa Cruz Biotechnologies, Santa Cruz, CA, USA), RANKL (rabbit polyclonal antibody from Santa Cruz Biotechnologies) and the loading control α-tubulin (rabbit polyclonal antibody from Abcam, Cambridge, MA, USA). Anti-tubulin was used at 0.4 μg/mL, while the others were used at 1 μg/mL. Goat anti-rabbit and Donkey anti-mouse fluorochrome-conjugated secondary antibodies were purchased from Licor Biosciences (Lincoln, NB, USA). The protein bands were detected by a Licor Odyssey BioImager and quantified by densitometry using corresponding software (Licor Biosciences). Each band of ALP or RANKL was normalized to its corresponding band of loading control. Cell lysates from untreated cells were loaded onto every gel. The results were expressed as percentages of the corresponding untreated control results.

Western blot analysis of endogenous CFTR protein was performed as described previously (Cholon et al., 2014 (link); Gentzsch et al., 2016 (link)). Briefly, whole-cell lysates of fully differentiated CF HBE cultures were prepared and then CFTR was immunoprecipitated. Samples were separated on 4–20% gradient SDS–polyacrylamide gel electrophoresis gels (Bio-Rad) and then transferred to nitrocellulose. Blots were probed with mouse monoclonal anti-CFTR antibodies and then with IRDye–goat anti-mouse immunoglobulin G (Molecular Probes). Anti-tubulin (LI-COR) was used as a loading control. Protein bands were visualized using an Odyssey Infrared Fluorescence Imaging System (LI-COR).