Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

Project description:We sequenced pollen DNA from Col x Ler F1 hybrid plants using Nanopore long-read sequencing and identified crossover sites with COmapper. To purify pollen grains from F1 hybrid plants and extract their genomic DNA, whole inflorescences of hybrid plants were collected and transferred to ice-cold 10% (w/v) sucrose in a 50-ml tubes. Flowers were homogenized with a blender (Tefal, BL3051KR), filtered through 80-μm nylon mesh, and centrifuged at 350g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 10% (w/v) sucrose, filtered through a 40-μm cell strainer, and centrifuged at 100g for 10 min at 4°C. The purified pellet was frozen in liquid nitrogen and ground with a mortar and pestle. The pellet powder was resuspended in CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA). Proteinase K (NEB, P8107S) and RNase A (20 mg/ml, Roche, 10109142001) were added to a final concentration of 80 U/ml and 20 μg/ml, respectively. To check pollen disruption, a 1-μl sample was diluted in a 1:10 ratio with 10% (w/v) sucrose and observed under a stereomicroscope (Leica, M165 FC). Then, 500 μl of the resuspended pellet was transferred to a 1.5-ml tube, to which an equal volume of 25:24:1 (v/v/v) phenol:chloroform:isoamylalcohol (Sigma, 77617) was added, and the mixture was homogenized by gentle manual shaking. The tube was centrifuged at 15,000g for 10 min at 4°C, and 400 μl of supernatant was transferred into a new tube, to which a Nanobind disk (Nanobind® Plant Nuclei Kit RT, PacBio, 102-302-000) was added. The tube containing the Nanobind disk was gently mixed, 400 μl isopropanol was added, and the tube was slowly inverted and incubated for 2 h at room temperature on a rotator set at 7 rpm. A magnetic rack (DynaMagTM-2, Invitrogen, 12321D) was used to separate the disk and the supernatant, and the supernatant was removed. PW1 buffer in the Nanobind Plant Nuclei Kit was used for washing the disk twice and the supernatant was removed completely by centrifugation. Then, 150 μl EB+ buffer was added onto the disc and gently tabbed and incubated overnight at room temperature. The supernatant was transferred to a new 1.5-ml tube and stored at −20°C. The DNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853) and integrity checked by gel electrophoresis. Nine micrograms of gDNA from pollen was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).

Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

			Action	DRS
	24NP0162_10.raw	Raw
	24NP0162_11.raw	Raw
	24NP0162_12.raw	Raw
	24NP0162_9.raw	Raw
	checksum.txt	Txt

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Functional enrichment analysis of differentially expressed proteins

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Dietary quercetagetin enhances antioxidant capacity and modulates intestinal microbiota in dexamethasone-challenged broilers.

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