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Hepatitis C virus induces HuR relocalization by coordinating PKC-δ and AMPK-α function

ABSTRACT: Huh7.5 cells were transfected with HCV-JFH1 RNAand harvested after 48h of transfection. Total proteins were extracted in IP lysis buffer (Pierce, Thermo Scientific) containing 1× halt-protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) with intermediate vortexing followed by mild sonication. The lysate was centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant was quantified by the BCA method (Thermo Fisher Scientific, USA). An equivalent amount (4 mg) of proteins from each condition was incubated with 3g of anti-HuR antibody for overnight at 4 °C. The immunocomplex was captured by using protein G Sepharose 4 fast flow beads (17-0618-01/ GE Healthcare). The immunoprecipitated complex was washed two times with IP lysis buffer and one time with mili-Q. Bound protein complexes were eluted in 50 µl SDS-PAGE 2X Laemmli sample buffer. The samples were resolved on 12% SDS–PAGE and stained with Coomassie stain (0.1% w/v). The gel was subjected to further in-gel mass spectrometry analysis.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte

SUBMITTER: TUSHAR KANTI MAITI

LAB HEAD: Dr. Tushar K Maiti

PROVIDER: PXD035903 | Pride | 2023-09-16

REPOSITORIES: Pride

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Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

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Hepatitis C virus induces HuR relocalization by coordinating PKC-δ and AMPK-α function

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