Biomax xar film

The protocol was largely as previously described [28 ]. Briefly, the detached epithelial cell lysates were mixed with 2X modified Laemmli Sample Buffer containing 8 M urea and incubated for 30 min at room temperature. Following centrifugation (2 min, 8,000 x g), samples of the supernatant (adjusted to 40–50 μg protein) were separated on a 6 % polyacrylamide gel. Immunoblot analysis was performed with an anti-CFTR antibody (NB100-92156, Novus Biologicals), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. Peroxidase activity was detected by incubating with Luminata Forte Western HRP substrate (Millipore) and exposure to X-ray film (Kodak® BioMax® XAR Film).

Radiolabeled probes for EMSAs were generated as described before (43 (link)). Uniformly labeled 126-nt blunt dsRNA was generated by in vitro transcription of T7 promoter-flanked PCR fragments using T7 RNA polymerase in the presence of α-³²P-[UTP]. After annealing of the two radiolabeled RNA strands, unincorporated nucleotides were removed using a G-25 Sephadex column (Roche) and dsRNA was purified from an 8% native polyacrylamide gel. Synthetic 21-nt siRNAs containing 2-nt 3′ overhangs and 19-nt blunt dsRNAs (43 (link)) were end-labeled with γ-³²P-[ATP] using T4 polynucleotide kinase (Roche) and purified on a G-25 Sephadex column.
EMSAs were performed as described previously (11 (link)). Briefly, radiolabeled 126-nt long dsRNA (5 ng), 19-nt dsRNA or siRNA duplexes (1 nM) were incubated with different concentrations of recombinant proteins for 1 h at room temperature. Long dsRNA and siRNA EMSAs were analyzed on 6% and 12% native polyacrylamide gels, respectively. Gels were exposed to a Kodak Biomax XAR film and radioactive signals were quantified with ImageJ software.

Western blot analysis was conducted as previously described [45 (link)]. Briefly, A549Pt and A549cisR cells were plated to approximately 70% confluency. Next day, cells were exposed to the designated treatment as described in the “Results” section. At the end of the incubation time, cells were lysed in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß-mercaptoethanol and boiled for approximately 5 minutes. Whole protein lysates were separated on a 10% polyacrylamide gel by SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA), then blocked for 1 hour in 10% milk in TBST (20 mM Tris, 137 mM NaCl, 0.1% Tween 20, pH 7.5) and probed with appropriate primary antibodies for 16 hours. Membranes were then incubated with HRP-conjugated secondary antibodies (1:5000) for one hour prior to signal development using ECL2. Kodak BioMax XAR film was used for chemiluminescence detection.

Cultures grown to early exponential phase in TYG broth were incubated with 100 μCi ³H-palmitic acid (PerkinElmer) for 2 h at 37°C. Cells were collected by centrifugation (4,000 x g, 20 min, 4°C), washed twice with PBS, and divided into two aliquots. One aliquot was treated with 2 mg/ml PK as described above. After PK treatment, both aliquots were lysed by sonication and centrifuged (18,000 x g, 20 min, 4°C) to remove unbroken cells and debris. The cleared lysates were then centrifuged at 100,000 x g for 45 min at 4°C, and the resulting membrane pellets were resuspended in PBS. Proteins were resolved by SDS-PAGE on 8–16% Tris-glycine minigels (Life Technologies). Gels were fixed, treated with Enlightening enhancer (Kodak) and dried. Radiolabeled proteins were then detected using Kodak Biomax XAR film.

GV-intact oocytes, ovulated eggs and two-cell embryos (10–15) were lysed in 4x LDS (lithium dodecyl sulfate) sample buffer with 10x reducing reagent (Life Technologies-Invitrogen), separated on 12% Bis-Tris precast gels, transferred to nitrocellulose membranes (Life Technologies-Invitrogen), blocked in 5% nonfat milk in TBS (Tris buffered saline, pH 7.4) with 0.1% Tween 20 (TBST) for 1 hr at room temperature, and then probed with 1:500–1:1,000 dilution of primary antibodies at 4°C overnight. On the following day, blots were incubated with a 1:10,000 dilution of secondary antibodies conjugated to HRP (horse radish peroxidase). Chemiluminescence was performed with ECL Plus (Piercenet) and signals were acquired by a Luminescent Image Analyzer LAS-3000 (Fujifilm, Valhalla, NY) or with BioMax XAR film (Kodak, Rochester, NY).

Ten µCi of ⁷⁵Se-selenite (University of Missouri, Columbia, MO, specific activity ∼1000 Ci/g) was administered to nursing dams intraperitoneally approximately 24 h prior to milk collection. Milk was collected in PBS and centrifuged at 16,000 g for 15 min to separate the lipid layer, the aqueous layer (whey), and the solids (curd). The whey fraction was transferred to a clean tube. A sample of whey was subjected to SDS-PAGE and the gel was stained with Coomassie blue. The stained 15% acrylamide gel was dried and exposed to Kodak BioMax XAR film.

To detect single-stranded regions of dsRNAs synthesized by Pol IV and RDR2, in vitro transcription reactions were performed as previously described (Singh et al., 2019 (link)). Oligonucleotides used for in vitro transcription reactions are listed in Supplementary file 1b. Resulting Pol IV-RDR2 dsRNAs were then subjected to the indicated amounts of S1 nuclease digestion for 10 min at 37°C using the manufacturer supplied S1 nuclease digestion buffer. Following this, RNAs were precipitated with 3 volumes of isopropanol, 1/10th volume of 3 M sodium acetate (pH 5.2), 20 µg of GlycoBlue (Thermo Fisher Scientific), and overnight incubation at –20°C. Precipitated RNAs were pelleted by centrifugation 16,000×g for 30 min, washed with 1 ml 70% ethanol, and resuspended in 2× RNA Loading Dye (NEB). Resuspended RNAs were incubated at 72°C for 5 min and loaded onto a 15% polyacrylamide 7 M urea gel. Following electrophoresis, gels were transferred to filter paper, vacuum dried, and subjected to autoradiography using BioMax XAR film (Kodak).
Dicing and capping of siRNAs generated from in vitro Pol IV-RDR2-DCL3 reactions were performed as previously described (Singh et al., 2019 (link)).