Rluc-ATP4A was expressed in vitro using the TnT SP6 Quick Coupled Transcription/Translation kit (Promega), based on transcription by the SP6 phage RNA polymerase and translation by a rabbit reticulocyte lysate cell-free expression system. Nluc-ATP4B was expressed in eukaryotic cells, using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA). In the Expi293 expression system, recombinant protein expression is achieved by high efficiency transfection of Expi293F, a derivative of HEK293 cells, adapted to growth in suspension in a defined composition, serum free medium. After 48 h of growth with agitation, transfected Expi293F cells were pelleted and lysed with passive lysis buffer (Thermo Fisher Scientific). Expression of recombinant antigens was assessed by quantification of luciferase activity in the lysates after the addition of Renilla luciferase assay system substrate or NanoGlow substrate (Promega), reconstituted according to the manufacturer instructions, for ATP4A and ATP4B, as appropriate. Luciferase activity was measured using a Berthold Centro xS960 luminometer (Berthold, Germany) and expressed in light units (LU) emitted over a time interval of 2 s. Recombinant antigen preparations were aliquoted and stored frozen at −80 °C.
Passive lysis buffer (plb)
Manufactured by Thermo Fisher Scientific
Sourced in United States
Passive lysis buffer is a solution designed for the gentle extraction and release of cellular contents, including proteins, from biological samples. It facilitates the lysis of cells while preserving the integrity of the target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Glomax 20 20 luminometer, by Promega (2 mentions)
Renilla luciferase assay system substrate, by Promega (1 mentions)
Nanoglow substrate, by Promega (1 mentions)
Centro xs960 luminometer, by Berthold Technologies (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
α actin, by Cell Signaling Technology (1 mentions)
Multiskan ascent photometer, by Thermo Fisher Scientific (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Veritas microplate luminometer, by Promega (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Dual luciferase kit, by Promega (1 mentions)
Prl tk renilla luciferase vector, by Promega (1 mentions)
X tremegene hp transfection reagent, by Roche (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Mouse anti ha antibody clone 16b12, by BioLegend (1 mentions)
12 protocols using passive lysis buffer (plb)
1
In Vitro and Eukaryotic Expression of ATP4A and ATP4B
2
Measuring Notch Signaling Activity
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Transfected and heat-shocked HeLa cells were washed with PBS and lysed with passive lysis buffer (Promega), which had been diluted to 1× with mq H2O. For measuring luciferase activity from cells expressing the 12×CSL luciferase reporter gene, 2 µl of sample was mixed with 18 µl of passive lysis buffer, and the luciferase activity was measured from triplicates with a luminometer (Thermo Scientific). The 12×CSL luciferase reporter has been described earlier [48 (link), 49 (link)]. For measuring β-galactosidase activity, a mixture of 10 µl of sample and 240 µl of ONPG (ortho-nitrophenyl-β-galactoside) buffer was incubated for 30 min at 37 °C, and the absorbance was measured from triplicates with a Multiskan Ascent photometer (Thermo Scientific) at 420 nm. Notch activity was calculated by relating the luciferase expression levels to the expression levels of β-galactosidase. To verify equal expression levels of Notch, lysates for immunoblotting were taken prior to adding passive lysis buffer. The lysates were immunoblotted against Notch with α-Notch1 C20 (Santa Cruz Technology), and α-actin (Cell Signaling) was used as a loading control. Values shown in the figures are statistically significant at ***P < 0.001 or *P < 0.05 as indicated. Values indicate the average of at three independent experiments.
3
Dual-luciferase Assay for CHCHD2 Promoter
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The HepG2 cells were cotransfected with 0.4 μg plasmid-constructed CHCHD2 promoter and 13 ng internal control plasmid phRL-TK using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. At 24 h post-transfection, the cells were harvested and lysed in 50 μl passive lysis buffer (Thermo Fisher Scientific). A fraction of the protein was subjected to a Dual-luciferase Reporter Assay System kit (Promega Corp.). The firefly luciferase activity and Renilla luciferase activity were measured sequentially using a Veritas Microplate Luminometer (Turner BioSystems, Inc., Sunnyvale, CA, USA). All transfections were performed in triplicate and the promoter activities were expressed as the mean ± standard deviation of three independent experiments.
4
IFNβ Promoter Activation Assay with PR8 NS1 Variants
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The plasmids expressing IFNβ-promoter (−125) firefly luciferase, Renilla Luciferase vector (pRL-TK; Promega), RIG-I and each PR8 NS1 (WT, G45R and AA) were co-transfected into 293 T cells grown in 24-well plate as previously described [45 (link)] with some modifications. Briefly, the indicated plasmids were transfected to the cells using XtremeGene HP transfection reagent (Roche). At 24 h post transfection (pt), the cells were lysed in passive lysis buffer (Invitrogen) and subjected for luciferase activity measurement using dual-luciferase kit following the manufacturer’s instruction (Promega). The firefly luciferase activity signals were normalized to the Renilla luciferase activity. The experiments were done in triplicate. NS1 expression levels in transfected cells were shown by western blot analysis (Additional file 2 : Fig. S2).
5
VEGF-A 3'UTR Regulation by miR-140
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The dual-luciferase reporter plasmid fused with the wild-type or mutant 3′-UTR segment of human VEGF-A was obtained from GenePharma (Shanghai, China). For verification of the regulation of VEGF-A by miR-140 mimics, a total of 7 × 104 HEk-293T cells stably transfected with -miR-140-5p mimics or miR-NC/N-control (scramble mimic) were seeded into 24-well plates. On the following day, the cells were transfected with 100 ng of the VEGF-A Wt or Mt 3′-UTR reporter plasmids, and 50 nM miR-140-5p mimics or scramble mimics with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After 2 days, cells were harvested with passive lysis buffer (Invitrogen) and the luciferase activity was calculated by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
6
Luciferase Assay for miRNA-139-5p Targeting
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100 ng of Wnt1 3′UTRs (forward, 5′-CCATGGGGCTCTGGGCGCTG-3′ and reverse, 5′-TTATAGATAAAAG-3′) containing miRNA-139-5p (forward, 5′-ACACTCCAGCTGGGTCTACAGTGCACGTG-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′) were amplified in psiCHECK™−2 Vectors (Promega Corporation, Madison, WI, USA) and co-transfected into cells using 1 ml Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h, cells were lysed in Passive Lysis Buffer (Invitrogen; Thermo Fisher Scientific, Inc.) and luciferase activity was measured using a DualGlo Luciferase Assay System (Promega Corporation).
7
HIF-2α Transcriptional Activity Assay
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End-point Luciferase assays were performed as previously described. 20, 26 Real-time Luciferase assays were performed on HEK 293T cells transfected with jetPRIME ® (Ozyme Polyplus). Expression vectors pcDNA3-HA-PHD2 were co-transfected with pcDNA3-HA-HIF-2α, pGL3 3xHRE-luciferase reporter plasmid and pCMV-HA-empty vector. Luciferase activity was measured over 24 h using the bioluminometer WSL-1565 Kronos HT ® (ATTO). Cells were harvested at different timepoints and lysed in passive lysis buffer (Invitrogen) for immunoblot detection with a mouse anti-HA antibody (clone 16B12, BioLegend).
8
Immunoblotting and Immunoprecipitation in LV
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Immunoblotting in LV homogenates was performed using primary antibodies raised against NOS enzymes, Cav-3, the Ca2+ ATPase (SERCA2A), the Na+-Ca2+ exchanger (NCX1), GAPDH (Santa Cruz Biotechnology), RyR2 (Thermo Fisher Scientific) and total and Ser16- phosphorylated phospholamban (PLB, Badrilla). For immunoprecipitation assays, LV were homogenized in IP buffer (1%Triton X-100, 150mM NaCl, 10mM Tris-HCl pH 7.4, 1mM EDTA, 1 mM EGTA and a protease inhibitor cocktail from Roche Diagnostics) and 350 µg of protein were precleared using 25 µl of Protein A/G–conjugated agarose. Samples were incubated overnight at 4 °C with primary antibodies at a final concentration of 10 µg/ml. Protein A/G –conjugated agarose was used to precipitate the primary antibodies for an additional 4 h, washed three times in IP buffer and then boiled in 60 µl of loading buffer. Samples were then subjected to SDS polyacrylamide gel electrophoresis and immunoblotting. The same membrane was first blotted for iNOS, then stripped and re-probed for eNOS. Similarly, we first blotted for phospho Ser16-PLB and then for total PLB, after stripping the membrane.
9
Protein Expression Analysis by Western Blotting
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Western blotting was performed as previously described [20] (link). Proteins from homogenized right ventricles were separated by 10% or 15% SDS-PAGE gels and then transferred onto nitrocellulose membranes, which were incubated overnight with mouse monoclonal antibodies for SERCA2a (1∶1000, Thermo Scientific, IL, USA), PLB (1∶1000, Thermo Scientific, IL, USA USA), phosphorylated PLB at serine 16 (1∶5000, Badrilla, UK) and NCX (1∶1000, Thermo Scientific, IL, USA). After washing, the membranes were incubated with anti-mouse (1∶5000, Stressgen, Victoria, Canada) immunoglobulin antibodies conjugated to horseradish peroxidase for 1 hour. After thorough washing, immunocomplexes were detected using an enhanced horseradish peroxidase/luminal chemiluminescence system (ECL, Amersham International, Little Chalfont, UK) and film (Hyperfilm ECL International). Signals on the immunoblot were quantified using the Image J computer program. Each membrane was reprobed to determine GAPDH expression using a mouse monoclonal antibody (1∶5000, Abcam Cambridge, MA, USA).
10
Cardiac Protein Expression Analysis
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Ventricles, n = 5⿿7/treatment/sex/diet, were homogenized in RIPA buffer (1% NP-40, 50 mM Tris (pH 7.4), 0.5% deoxycholate, 159 mM NaCl, 0.1% SDS, 10 mM sodium metabisulfite, 1 mM sodium vanadate, proteinase inhibitor cocktail, PhosSTOP (Roche, Indianapolis, IN), and 1 mM PMSF). Protein was measured using the Bradford Protein Determination Assay (BioRad, Hercules, CA) as per the manufacturer⿿s instructions.
Protein expression was measured using standard immunoblotting methods. Primary antibodies to the cardiac calcium homeostasis proteins sodium calcium exchanger-1 (NCX1, Abcam Inc, Cambridge, MA ab6495); sarcoplasmic calcium ATPase 2a (SERCA2a, Santa Cruz Biotechnology, Santa Cruz, CA, sc-8095); cardiac calsequestrin 2 (CASQ2, Abcam Inc, Cambridge, MA, ab626662); phospholamban (PLB, Thermo Scientific, Nepean, ONT, MA3-922); and phospho-serine 16-specific PLB (pS16-PLB, Millipore, Temecula, CA, 07-052 1) were obtained. Secondary antibodies complexed to horseradish peroxidase and chemiluminescent detection kits were obtained from Pierce Chemical Co., (Rockford, IL). Several exposures from each membrane were collected onto X-ray film. After immunoblotting, membranes were washed, permanently stained with Coomassie Brilliant Blue, destained and scanned. The bands on the X-ray film and stained membrane were scanned and quantitated using Image J software.
Protein expression was measured using standard immunoblotting methods. Primary antibodies to the cardiac calcium homeostasis proteins sodium calcium exchanger-1 (NCX1, Abcam Inc, Cambridge, MA ab6495); sarcoplasmic calcium ATPase 2a (SERCA2a, Santa Cruz Biotechnology, Santa Cruz, CA, sc-8095); cardiac calsequestrin 2 (CASQ2, Abcam Inc, Cambridge, MA, ab626662); phospholamban (PLB, Thermo Scientific, Nepean, ONT, MA3-922); and phospho-serine 16-specific PLB (pS16-PLB, Millipore, Temecula, CA, 07-052 1) were obtained. Secondary antibodies complexed to horseradish peroxidase and chemiluminescent detection kits were obtained from Pierce Chemical Co., (Rockford, IL). Several exposures from each membrane were collected onto X-ray film. After immunoblotting, membranes were washed, permanently stained with Coomassie Brilliant Blue, destained and scanned. The bands on the X-ray film and stained membrane were scanned and quantitated using Image J software.
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