All samples were incubated at 95 °C for 10 min, separated on a 4–12% polyacrylamide Bis-Tris protein gel (NP0322BOX, Thermo), and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The membranes were blocked in 10% w/v milk (1706404, Bio-Rad). The blots were probed with 1/1000 v/v EIF2S1 antibody (AHO0802, Invitrogen) or 1/500 v/v Phospho-EIF2S1 (Ser51) antibody (MA5-15133, Invitrogen), followed by 1/10000 v/v sheep anti-mouse IgG (NA931V, Cytiva) or 1/10000 v/v donkey anti-rabbit IgG conjugated to horseradish peroxidase (NA934V, Cytiva). The blots were visualized with ECL Prime Western Blotting Detection Reagents (RPN2232, Amersham) using a Bio-Rad Chemi-Doc MP.
Na931v
Manufactured by Cytiva
Sourced in United States, Italy, United Kingdom
The NA931V is a laboratory centrifuge produced by Cytiva. It is designed to separate various biological samples, such as cells, proteins, or nucleic acids, based on their density. The centrifuge can reach a maximum speed of 14,000 revolutions per minute and can accommodate rotor configurations that allow for the processing of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Na934v, by Cytiva (16 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ipvh00010, by Merck Group (2 mentions)
Precision red advanced protein assay reagent, by Cytoskeleton (2 mentions)
Luminata forte western horseradish peroxidase substrate, by Merck Group (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
α tap cab1001, by Thermo Fisher Scientific (2 mentions)
α ha clone 12ca5, by Roche (2 mentions)
Gel doc 2000, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Westernbright ecl, by Advansta (2 mentions)
Ab654, by Abcam (2 mentions)
Ab113474, by Abcam (2 mentions)
Mmv00, by R&D Systems (2 mentions)
Nb300 666, by Abcam (2 mentions)
Ab652, by Merck Group (2 mentions)
Ab1344, by Abcam (2 mentions)
Nb100 134, by Novus Biologicals (2 mentions)
Fibrillarin, by Cell Signaling Technology (2 mentions)
24 protocols using na931v
1
Western Blot Analysis of EIF2S1 and Phospho-EIF2S1
2
Transfection and Reporter Assay in HEK293T Cells
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HEK293T cells were plated at a density of 5 × 105 cells in 2 mL per well in 6-well plates and after 22–24 h, the calcium phosphate method was used to transfect the cells with 200 ng of pCSK-LacZ (Condie et al., 1990 (link)), with or without 1500 ng of Igκ2-IFN-LUC (Pomerantz et al., 2002 (link)), with or without the indicated amount of the pcDNA3 myc-CARD11 constructs, and the empty pcDNA3 vector to achieve 3000 ng of total DNA per condition. At 24 h post transfection, the media was replaced with 2 mL fresh complete DMEM and at 40–44 h post transfection, the cells were harvested in 400 μL 1× Reporter Lysis Buffer followed by incubation on ice for 10 min and centrifugation at 13,000 rpm for 10 min at 4°C. The chemiluminescent β-gal reporter gene assay (Roche, 11758241001) was performed using 2 μL of lysate according to the manufacturer's instructions using a Berthold Technologies TriStarLB 941 luminometer, integrating for 10 s after a 2 s delay. The measured β-gal activity was used to normalize for transfection efficiency and extract recovery and the normalized lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by western blotting with the mouse anti-myc primary antibody (Santa Cruz, sc-40) and the sheep anti-mouse (Cytiva, NA931V) secondary antibody to determine relative expression. Western blots represent one of at least two replicate experiments.
3
Immunostaining of APOA1 and Flot-1
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The following antibodies were used for immunostaining: mouse monoclonal anti‐APOA1 (1:100, B10, Santa Cruz Biotechnology, Dallas, Texas, USA), anti‐Flot‐1 (1:1000, 610820, BD Biosciences, Franklin Lakes, New Jersey, USA), sheep anti‐mouse horseradish peroxidase‐linked antibody (1:4000, NA931V, Cytiva, Massachusetts, USA) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:3000, NA934V, Cytiva, Massachusetts, USA).
4
Western Blot Antibody Detection
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5
Protein Expression Analysis Protocol
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Cells were lysed with lysis buffer (a cocktail of 20 mM Tris-HCl pH 8.0, 60 mM NaCl, 0.2% glycerol, 0.02% NP-40, 0.04 mM EDTA, 1 mM PMSF, and 1.52 mg/mL protease inhibitor). Proteins were analyzed with SDS-PAGE gel and transferred onto a polyvinyl difluoride membrane (PVDF) (Millipore, Temecula, CA, USA). The PVDF membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies used were anti-Kla (PTM BIO, PTM-1401,1:1000), anti-β-Tubulin (Abmart, M20005H, 1:5000, Shanghai, China), anti-Ldha (Cell Signaling Technology, 2012S, 1:1000, Danvers, MA, USA), anti-Nanog (Bethyl Laboratories, A300-397A, 1:2000), anti-Oct4 (Santa Cruz Biotechnology, sc-5279, 1:1000, Dallas, TX, USA), anti-Esrrb (Perseus Proteomics, H6705-00, 1:1000, Tokyo, Japan), and anti-FLAG (Sigma-Aldrich, F1804, 1:3000). After washing with TBS 3 times, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. The secondary antibodies used were donkey anti-Rabbit (Cytiva, NA934V, 1:5000, Marlborough, MA, USA), goat anti-Rabbit (Santa Cruz, sc-2354, 1:1000), and sheep anti-Mouse (Cytiva, NA931V, 1:5000, Marlborough, MA, USA). ECL solution (Shanghai Epizyme Biomedical Technology Co., Ltd., Shanghai, China) was added, and signals were detected using an automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China).
6
TRPA1 Protein Expression Analysis
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Proteins (10 µg) were mixed with 2 × Laemmli buffer supplemented with 2-mercaptoethanol, and denatured for 5 min at 95 °C. Samples were loaded in a 4–20% Mini-PROTEAN TGX Precast Gel (Bio-Rad). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad). Membranes were blocked with TBST (0.2% Tween-20 diluted in Tris-buffered saline) containing 5% Blocking-One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies for anti-TRPA1 (rabbit, Polyclonal, 1:500; NB110-40763, Novus Biologicals, LLC, Centennial, CO, USA), and anti-β-actin antibody (mouse monoclonal, 1:200; sc-69879; Santa Cruz, San Diego, CA, USA). After washing with TBST 4 times for 5 min, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for anti-rabbit IgG or anti-mouse IgG (1:2000, NA934V or NA931V; Cytiva, Marlborough, MA, USA) 2 h at room temperature. The bands were visualized using an image analyzer (Amersham Image Quant 800, Cytiva) after reaction in Western Lightning ELC Pro (PerkinElmer, Waltham, MA, USA) or Immobilon (Millipore, Burlington, MA, USA). The band intensity was quantified using ImageJ and normalized to β-actin expression.
7
TMEM230 Protein Expression Analysis in HUVECs
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HUVECs were lysed on ice using Laemmli buffer, as previously described [5 (link)]. Briefly, 20 µg of total protein for each sample was mixed with a 6x loading dye buffer and loaded onto 10% SDS denaturing poly-acrylamide gels. After transferring proteins to a PVDF membrane (10600021 Euroclone), the membrane was blocked with 5% fat-dried milk (EMR180001 Euroclone) and incubated with primary polyclonal rabbit anti-TMEM230 (1:2500, 21466-1-AP, Proteintech, Rosemont, IL, USA,) and polyclonal goat anti-Lamin A/C (sc 376248 Santa Cruz Biotechnology, Dallas, TX, USA) used as endogenous control at concentration 1:7500. Donkey anti-rabbit (1:20,000, NA934V, Amersham, Cologno Monzese, Mi, Italy) and sheep anti-mouse (1:10,000, NA931V Amersham) were used as secondary antibodies.
8
Protein Extraction and Western Blot Analysis
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Cells were lysed with RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl, pH 7.5) containing protease inhibitors. The protein concentration was determined using DC protein assay (Bio-Rad, Hercules, CA, USA), and equal amounts of protein were separated on 6–12% SDS-PAGE Gels and transferred to PVDF membrane (Invitrogen, Waltham, MA, USA). Nonspecific protein binding was blocked using either 3% Bovine Serum Albumin (BSA) or 5% powdered milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h followed by incubation with primary antibodies diluted in the blocking solution, overnight at 4 °C. The following morning, the membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, NA931V, and anti-rabbit, NA934V, Amersham) for 1 h at room temperature. Membranes were then washed and imaged. Images were developed using the Immobilon Western Kit (Millipore, Burlington, MA, USA) and detected on a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA), and Bio-rad (version S.0.2.3.0, Rochester, NY, USA) was used to quantify protein content. GAPDH (1:10,000, no. ab9484, Abcam, Cambridge, United Kingdom) was used as a loading control. (Table 2 ).
9
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amount of protein lysate was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore) using fast Turbo Transfer (Bio-rad). After blocking in 5% skimmed milk for 1 h, the appropriate primary antibody was added: anti-ADAR2 (RED1) (1:1000, ab64830, Abcam), anti-SRp30c(SRSF9) (1:1000, sc-134036, Santa Cruz), anti-FLAG (1:2000, Clone M2, Sigma), anti-cMyc (1:1000, sc-789, Santa Cruz), anti-V5 (1:2000, ab9116, Abcam) or anti-β-actin (1:1000, clone C4, sc-47778, Santa Cruz). Primary antibodies were incubated overnight in the cold room. After washing with phosphate-buffered saline (PBS)/0.2% Tween-20 (PBST), a secondary antibody, namely horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000, NA934V, Amersham) or HRP-conjugated anti-mouse IgG (1:5000, NA931V, Amersham), was added for 1 h. After washing with PBST, signals were detected using the WesternBright Sirius Chemiluminescent HRP Substrate (Advansta).
10
Quantitative Western Blot Analysis of Protein Abundance
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The samples derived from human keratinocytes and mouse skin tissues were lysed with RIPA lysis buffer (9806, Cell Signaling Technology). Protein concentrations were determined using Precision Red Advanced Protein Assay reagent (ADV02, Cytoskeleton), and equal amounts of total protein were subjected to electrophoresis with 8%–15% SDS-PAGE gels followed by transfer to PVDF membranes (IPVH00010, Merck Millipore, Burlington, Massachusetts, USA). The membranes were then blocked in ImmunoBlock buffer for 1 hour at room temperature followed by overnight incubation at 4°C with primary antibodies according to the manufacturer’s instructions. Labeling of the primary antibodies was detected using sheep anti-rabbit or sheep anti-mouse antibodies conjugated to horseradish peroxidase (NA934 V and NA931 V, respectively; Amersham Biosciences), developed with the Luminata Forte Western horseradish peroxidase substrate (WBLUF0100, Merck Millipore, Billerica, Massachusetts, USA), and then imaged using Fujifilm LAS-4000 Plus. ImageJ was used for quantification of the band intensity in the images.
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