Visionworks ls software

For lipid analyses, cell suspensions were extracted with chloroform–methanol (1:1). High-performance thin layer chromatography was performed as previously described with modification to a single solvent system [petroleum ether (b.p. 60–70 °C)-ethyl ether-acetic acid (45:5:0.5)]^{59 (link)}. The images were analyzed using UVP Vision Works LS software by calculating the area under the curve after lane-specific straight line background correction. A mixture of the following standard lipids was co-chromatographed: cholesterol, trioleate glyceride, cholesteryl palmitate, and oleic acid. Preliminary analyses were completed to establish the linearity of detection for each lipid class to ensure that lipids did not exceed the linear range for quantitation. For every plate of cellular lipids, five lanes of varying amounts of lipid standards were simultaneously run to generate standard curves for quantitation. The amount of each cellular lipid was expressed as µg lipid/mg cell protein or µg lipid/mg initial tissue mass. The protocol was adapted from^{60 (link)}.

Cells were lysed in RIPA buffer (Enzo Life Sciences) supplemented with protease inhibitors and phosphatase inhibitor cocktail (Roche). The cell lysates were clarified by centrifugation at 20,000 × g for 15 min and followed by protein quantification with the BCA assay kit (Thermo Fisher Scientific). The cell lysates with similar protein concentrations were subsequently applied to Bis-Tris gels for protein separation, and the proteins were transferred from gels to polyvinylidene difluoride (PVDF) membrane by dry transfer (Thermo Fisher Scientific, iBlot 2 Gel Transfer Device). Immunoblot analysis was performed with the indicated antibodies, and the chemiluminescence signal was visualized with Luminata Forte Western HRP substrate (EMD Millipore) in the BioSpectrum system (UVP, LLC). The chemiluminescence intensities of the bands were quantified in the VisionWorks LS software (UVP, LLC).

Expression analysis was performed by a protocol that, using an optimized set of primers, allows the display of any possible anomalous splicing of CFTR mRNA, as previously described [37 (link)]. Briefly, starting from nasal brushing of patients, RNA was extracted by the RNeasy mini kit (Qiagen), reverse transcribed and amplified by RT-PCR (Bio-Rad, Hercules, CA, USA) producing 7 amplicons spanning the entire CFTR mRNA. All the 7 cDNA amplicons were extracted from agarose and individually sequenced as described above. The primers specifically used for the analysis of exon 10 splicing are reported in Table 3. In particular, one forward primers (F4, located in exon 9) and two different reverse primers (R4, located in exon 12 and R5, located in exon 11) were used. All cDNA amplicons, spliced and unspliced, were recovered from agarose gel and forward sequenced by F4 primer and reverse sequenced by R4 or R5 primers. This allowed the evaluation of exon 10 splicing and of the presence of p.Phe508del pathogenic variant, as well as of their segregation on different alleles also at cDNA (RNA) level. Gel electrophoresis runs were scanned by a CCD camera (VisiDoc-It; UVP, Cambridge, UK) and analyzed on the VisionWorks LS software version 6.7.3 (UVP) for densitometric assays. Expression analysis results were confirmed by Real Time PCR.

Cells were harvested and cell lysate were prepared by incubating the cells in RIPA (radio-immunoprecipitation assay) buffer containing protease inhibitor cocktail (10 µl/ml) (Roche Molecular Biochemicals, Mannheim, Germany) for 30 minutes on ice. Total protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Cell lysate (20 µg total protein per sample) were subjected to SDS-PAGE followed by transferring to PVDF membrane. The membrane was incubated for 1 hour in blocking buffer, probed with the primary antibody for 1 hour at room temperature, and then washed twice with blocking buffer without milk. The membrane was then incubated with the appropriate secondary antibody for 1 hour at room temperature. After washing with blocking buffer, the protein bands were visualized using the Western Lightning Plus-ECL Reagent (Perkin Elmer, Waltham, MA, USA). Images of the blots were captured using the BioSpectrumAC Imaging System (UVP, Upland, CA, USA). The band intensity was quantified using UVP Visionworks LS software.
The anti-Endothelin 1 antibody was purchased from Abcam (Cambridge, MA, USA) and the anti-β actin antibody from Sigma-Aldrich (St. Louis, MO, USA). The mouse HRP-conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

To extract DNA, equal numbers of FACS sorted peritoneal cells were resuspended in 25 mM NaOH, incubated at 95°C for 15 minutes, and neutralized with 40 mM Tris-HCl. DNA was amplified with GoTaq DNA Polymerase (Promega) with the following primers: Il4rα wild-type: F - 5′-GTACAGCGCACATTGTTTTT-3′, R - 5′-CTCGGCGCACTGACCCATCT-3′; Il4rα knockout: F - 5′-GGCTGCCCTGGAATAACC-3′, R - 5′-CCTTTGAGAACTGCGGGCT-3′. Gel was imaged and band intensity quantified with BioSpectrum set up with VisionWorksLS software (UVP; Upland, CA).