HA-S6K1ki wild type and mutants were cloned into the pcDNA3.1(+) vector. HEK-293F cells (Invitrogen) were maintained in DMEM medium with 10% fetal bovine serum (Sigma) at 37°C and 5% CO2. For transfection, cells were seeded into 6-well tissue culture plates, cultured to 70% confluence and exchanged into fresh media one hour prior to transfection. Cells were transfected with 2 μg each of HA-S6K1ki wild type or mutant plasmids using Lipofectamine 2000 (Invitrogen). 48 hours post transfection, cells were lysed in 50 mM Tris-HCl, 150 mM NaCl, 0.5 mM TCEP, 1% Triton-X100, 2 mM EDTA, 50 mM NaF, 10 mM β-glycerolphosphate and 10 mM Na-pyrophosphate, pH 7.5, and 1 tablet each of cOmplete protease inhibitor and PhosSTOP cocktail (Roche). Whole cell extract (W.C.E.) were adjusted to 2 mg ml−1 with lysis buffer and NuPAGE LDS sample loading buffer (Invitrogen), and boiled for 5 minutes. 20 μg W.C.E. were loaded on gel for immunoblotting with anti-HA antibody (Santa Cruz, SC805) or anti-phospho-S6K1 (T389) antibody (Cell signaling, #9205). The immunoblots were quantified by normalizing the anti-phospho-S6K1 signal to the anti-HA signal of each reaction.
Nupage lds sample loading buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE LDS sample loading buffer is a sample preparation reagent used in gel electrophoresis. It is designed to be used with NuPAGE electrophoresis systems. The buffer helps denature and prepare protein samples for separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Hek293f cell, by Thermo Fisher Scientific (2 mentions)
Anti phospho s6k1 t389 antibody, by Cell Signaling Technology (2 mentions)
Phosstop cocktail, by Roche (2 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Rabbit anti tak1, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho smad1 5 8, by Cell Signaling Technology (2 mentions)
Rabbit anti smad 1 5 8, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho tak1, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti rabbit 1 1000, by GE Healthcare (2 mentions)
Supersignal west pico chemiluminescenct substrate kit, by Thermo Fisher Scientific (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Complete mini, by Roche (2 mentions)
Ab129068, by Abcam (2 mentions)
11 protocols using nupage lds sample loading buffer
1
HA-S6K1 Kinase Mutant Analysis
2
Bmp7 Signaling Pathway Activation Assay
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Protein extracts were prepared from E7 BPs treated with Bmp7 for 15 or 30 minutes at room temperature. Tissue was treated with RIPA buffer (SIGMA Aldrich) containing protease (Complete mini; Roche) and phosphatase (SIGMA Aldrich) inhibitor tabletsat 4 °C. Tissue was left on ice for 2 hrs with periodic vortexing. Tissue lysates were subsequently denatured at 85 °C for 5 minutes with NuPAGE LDS sample loading buffer (Invitrogen) and β-mercaptoethanol (SIGMA Aldrich). Proteins were then separated using SDS-PAGE on a 4-12% gradient Bis-Tris gel (Invitrogen) and subsequently transferred to nitrocellulose membranes (Invitrogen) over night at 4 °C. The primary antibodies and dilutions used were as follows: Rabbit anti-Tak1 (1:1000, Cell Signaling), Rabbit anti-phospho-Tak1 (1:1000, Cell Signaling), rabbit anti-Smad 1/5/8 (1:1000, Cell Signaling), rabbit anti-phospho Smad 1/5/8 (1:1000, Cell Signaling), mouse anti-β-Actin (1:5000, Sigma Aldrich). Horseradish peroxidase-conjugated anti-rabbit (1:1000) or anti-mouse (1:5000) IgGs were used as secondary antibodies (GE Healthcare). Specific bands were visualized by chemiluminescence using the SuperSignal® West Pico Chemiluminescenct Substrate kit (Thermo Scientific).
3
Immunoblotting of Brain Protein Markers
4
HA-S6K1 Kinase Mutant Analysis
5
Western Blot Analysis of Protein Extracts
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Cells were washed in PBS, lysed directly in RIPA buffer (ThermoFisher) and protein extracts were quantitated using the Bicinchoninic acid assay (ThermoFisher) against a BSA standard curve. Extracts were made up in 4× NuPAGE LDS sample loading buffer (Invitrogen) supplemented with 100 mM dithiothreitol (Sigma-Aldrich), and incubated at 95°C for 10 min. Lysates (50–100 μg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on NuPAGE 3–8% Tris–acetate gels (Invitrogen) in NuPAGE Tris–acetate running buffer (Invitrogen), or NuPAGE 4–12% Bis–Tris gels (Invitrogen) in NuPAGE MOPS SDS running buffer (Invitrogen). Gels were wet-transferred in 1× NuPAGE Transfer Buffer (Invitrogen), 20% ethanol and 0.05% SDS to nitrocellulose membranes (Millipore). 5% BSA/Tris-buffered saline + 0.01% Tween-20 (TBST) or 5% milk/TBST was used for blocking and incubation steps. Membranes were probed overnight at 4°C with indicated antibodies. The membrane was washed thrice for 5 min with TBST and incubated with HRP- or fluorescent dye-conjugated secondary antibodies for 1 h at room temperature. After four 5 min washes with TBST, HRP signals were detected with ECL detection reagent (BioRad) and imaged on an Amersham Imager 600RGB and fluorescence was imaged directly on a LI-COR Odyssey M Imager. Antibodies used in this study are described in Supplementary Table 4 .
6
Western Blot Analysis of Protein Extracts
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Tissue protein extracts were prepared from frozen samples using 1% SDS (Sigma) in 50mM pH 8.0 Tris buffer (Sigma) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Pierce). Samples were sonicated on ice. Bicinchoninic acid assay (Pierce) was used to quantify protein concentrations. NuPAGE® LDS sample loading buffer plus sample reducing agent (Invitrogen) were added to lysates. Samples were loaded in Novex 4-12% Bis-Tris gels in MES buffer (Invitrogen) for electrophoresis separation, and transferred to 0.2 μM Immobilon Psq membranes (Millipore) for western blot analysis. 5% milk in Tris buffered Saline plus 0.1% Tween-20 (TBS-T) (Fischer Scientific) was used as a blocking buffer as well as antibody diluent. Primary antibodies to detect KISS1R (Alomone Labs), KSR1 (Santa Cruz Biotechnologies), CAMK1 (Abnova), SSPN (Santa Cruz Biotechnologies), and β-actin (Sigma-Aldrich) were used. All secondary species-specific horseradish peroxidase-labeled antibodies were purchased from Jackson Immunoresearch and Supersignal chemiluminescent kit (Pierce) was used to perform detection.
7
Bmp7 Signaling Pathway Activation Assay
8
Immunoblotting of Brain Protein Markers
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The cortex from E4FAD mouse brains was homogenized in Eppendorf tubes (kept on ice), using 300 ul of RIPA buffer (Thermo Fischer Scientific) with freshly added protease and phosphatase inhibitor cocktails. The samples were placed on an orbital shaker at 4°C overnight and spun the next day at 12000 rpm for 20 min at 4°C. The supernatant from each sample was collected and the protein concentration was assayed using BSA as working standard. Equal amounts of protein (40 μg) were heat-denaturized in NuPAGE LDS sample- loading buffer (Invitrogen) for 5 min at 95°C, resolved by SDS-PAGE and transferred to PVDF membranes (Sigma-Aldrich, MO, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk (NOX2, Aβ42) and Tris-buffered saline (TBS) containing 0.05% Tween and 2% BSA (Vinculin) and then incubated overnight with antibodies directed against NOX2 (Rabbit monoclonal, 1:5000, Abcam, ab129068), Aβ42 (Rabbit monoclonal, 1:500, Abcam, ab201060) or Vinculin (Mouse monoclonal, 1:1000, Millipore, MAB3574). Peroxidase conjugated IgG was used as secondary antibody. Membrane-bound immune complexes were detected by HyGLO™ Chemiluminescent HRP Detection Reagent (Denville Scientific). Protein loading was normalized according to Vinculin expression. Quantification was performed by densitometric analysis using Imagelab 6.0 software (Bio-Rad).
9
Western Blot Analysis of Viral Proteins
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Cells were lysed with RIPA buffer and with NuPAGE LDS sample loading buffer (Invitrogen) for 20 min and heated at 95% for 3 min before loading total protein samples (∼10 µl) onto a 15-well precast Express PAGE gel (Genescript). The proteins were separated by electrophoresis at 120 V for 1 h and transferred to PDVF membrane (GE Healthcare) for 1.5 h at 45 V using a wet transfer apparatus (BioRad). Membranes were blocked for 1 h in 5% non-fat milk (BioRad) and then probed for 1 h at room temperature with gentle rocking with primary antibodies diluted in 5% non-fat milk. The membranes were washed 3 times with TBST (at least 5 min each time) and incubated for 1 h with secondary antibodies diluted in PBS. After a final wash with TBST, the blots were developed by LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) and integrated intensity of each band was used for calculating the ratios. Ratios of structural (or reporter β-gal activity) vs non-structural proteins observed during WT virus infections or WT replicon RNA replication were set to 1 and this was used as the reference for calculating corresponding ratios in PRF-deficient virus or PRF-deficient replicon replication.
10
Protein Enrichment and Analysis via IGF
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Probe 2-treated and AzTB-labelled cell lysate samples to be analysed by IGF were incubated with magnetic Dynabeads (Streptavidin MyOne) (Thermo Fisher Scientific). Beads were prewashed with three volumes of 0.2% SDS, with rotary mixing for 3 min at RT. The samples were added to the beads and moderately shaken for 2 h at RT. The supernatant was removed, retaining an aliquot for SDS-PAGE analysis. The beads were then washed with three volumes of 0.2% SDS. Enriched proteins were eluted by boiling with 2% (v/v) 2-mercaptoethanol-containing NuPAGE LDS sample loading buffer (Thermo Fisher Scientific) at 95°C for 5 min. The samples were separated by gel electrophoresis.
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