Denatured biopsy specimens and cultured cell lysates were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk in PBS then incubated with mouse anti-HSV-1 ICP8 antibody (ab20194, Abcam, Cambridge, UK). Membranes were washed extensively and incubated with anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (NA9310V, GE Healthcare Biosciences, Piscataway, New Jersey, USA). Membranes were again washed extensively, incubated in Pierce ECL Western Blotting Substrate (Thermo Scientific) according to the manufacturer’s instructions, and visualized via autoradiography. To ensure adequate sample had been loaded for antibody detection, membranes were stripped and re-probed with anti-β-actin primary antibody (sc47778, Santa Cruz Biotechnology, Dallas, Texas, USA) and anti-mouse IgG secondary antibody (NA9310V, GE Healthcare).
Na9310v
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The NA9310V is a laboratory equipment device produced by GE Healthcare. It serves as a core function of performing essential tasks within the laboratory environment. The details and specific intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
Na9340v, by GE Healthcare (7 mentions)
Haf016, by R&D Systems (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Cellulose membranes, by GE Healthcare (2 mentions)
Ecl plus western blotting detection system, by GE Healthcare (2 mentions)
A5441, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amersham hybond c extra, by GE Healthcare (1 mentions)
A3853, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Phosstop phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
Ab49900, by Abcam (1 mentions)
Nupage lds reducing sample buffer, by Thermo Fisher Scientific (1 mentions)
Red ponceau s staining, by Merck Group (1 mentions)
Nb120 6588, by Novus Biologicals (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Ab54797, by Abcam (1 mentions)
20 protocols using na9310v
1
Western Blot Analysis of HSV-1 ICP8
2
Western Blot Analysis of Protein Targets
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Cells or small tissue slices were lysed on ice in RIPA buffer (50 mM HEPES, 10 mM NaCl2, 1% NP-40, 0.1% SDS and 1% Triton X-100) supplemented with cOmplete Protease Inhibitor Cocktail Tablets and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche, Mannheim, Germany) and 30 μg of the resulting proteins were separated on 10% SDS-PAGE then transferred to Amersham Hybond-C Extra (GE Healthcare, Solingen, Germany) membranes. Membranes were blocked in 5% milk powder in TBS-T0.1 then incubated with primary antibodies against Brd4 (1:200; #sc48772; Santa Cruz, Heidelberg, Germany), E2f1 (1:1000; #AF4825; R&D Systems, Minneapolis, MN, USA) or MYCN (1:1000; #9405; Cell Signaling, Frankfurt am Main, Germany) and Actin (1:2,000; #A3853; Sigma-Aldrich, Taufkirchen, Germany) or Gapdh (1:2000; #MAB374; Millipore, Darmstadt, Germany) as a loading control. After washing twice with TBS-T0.1, membranes were incubated 1h at room temperature with horseradish peroxidase-conjugated secondary antibodies against mouse IgG (1:2000; #NA9310V; GE Healthcare), rabbit-IgG (1:2000; #NA9340V; GE Healthcare) or sheep IgG (1:2000; #HAF016; R&D Systems). Protein detection and visualization were performed as described previously.30 (link)
3
Western Blot Analysis of ECM Proteins
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Five μg of proteins from control or CNV stromas were re-suspended in NuPAGE LDS reducing Sample Buffer (Thermo Fisher), resolved on NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher) and electro-transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK) for Western blot (WB) analysis. Protein transfer was evaluated by red Ponceau S staining (Sigma-Aldrich). Membranes were blocked in a Tris buffered solution (TBS) 5% milk, 0.1% Tween 20 and incubated overnight with primary antibodies: mouse anti-human decorin (MAB143), rabbit anti-human lumican and anti-human collagen-VI α1 (NBP1-87726 and NB120-6588, Novus Biologicals) at 4 °C under gentle shaking. Subsequently, membranes were incubated at RT for 1 h with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (NA9310V and NA9340V respectively, Ge Healthcare) followed by chemiluminescence reaction performed with ECL detection reagent (Ge Healthcare) and film exposure. The protein band optical density was finally measured with the UVITEC imaging system. Expression of β-actin revealed with an HRP conjugated mouse monoclonal antibody (ab49900, Abcam) was used as loading control.
4
Western Blotting Analysis of DPD Protein
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Cells and tumor tissues were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP40). For tissue lysis, 0.1% SDS was added with protease inhibitor cocktail set 1 (Fujifilm). Protein samples were separated by SDS-PAGE gel and transferred onto PVDF membranes. After 30 min blocking with Blocking-One (Nacalai Tesque), membranes were incubated with primary antibody overnight at 4 °C. The following antibodies were used: anti-DPD antibody (1:10,000, ab54797; Abcam), anti-GAPDH antibody (1:10,000, 14C10; Cell Signaling Technology, Danvers, MA, USA), and anti-α-tubulin antibody (1:10,000, PM054; MBL, Tokyo, Japan). Then membranes were incubated with secondary antibody (anti-mouse IgG, 1:20,000 [NA9310V, GE Healthcare]; anti-rabbit IgG, 1:20,000 [Jackson ImmunoResearch, West Grove, PA, USA]) for 1 h at room temperature. Each antibody was diluted in 1 × PBST. ECL substrates (Clarity Western ECL substrate; Bio-Rad) were used for signal detection with an electronically cooled charge-coupled device camera (LAS4000; GE Healthcare). Quantification of protein expression was performed with imageQuant TL (GE Healthcare)30 (link). As internal controls, GAPDH or α-tubulin was used for cells, and total protein was used for patients’ tissue samples. Total protein was detected by staining membranes with CBB-R (CBB Stain One Super, 11642-31; Nacalai Tesque) for 15 min.
5
Quantifying Amyloid Fibril Protein Levels
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Amyloid fibrils were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gels.
After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB). Another set
of electrophoresed gels was transferred onto PVDF membranes (Millipore, Billerica, MA,
USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) supplemented with
0.1% Tween 20 (PBST). The membranes were incubated with anti-mouse SAA1 goat antibody
(1:5,000) (AF2948, R&D Systems, Minneapolis, MN, USA), anti-bovine SAA1 monoclonal
antibody, 25BF12 (1:1,600) [29 (link)], which was purified
from the culture supernatant of hybridoma cells by protein A-sepharose (GE Healthcare), or
anti-chicken SAA1 mouse antibody (1:1,600) [22 (link)] in
1% skim milk in PBST at room temperature for 1 h. Membranes were washed three times with
PBST and incubated with anti-goat IgG donkey antibody F (ab’)2 conjugated with
horseradish peroxidase (HRP) (1:1,000) (SC3851, Santa Cruz Biotechnology, Dallas, TX,
USA), anti-mouse IgG F (ab’)2 conjugated with HRP (1:1,600)
(NA9310V, GE Healthcare, Buckinghamshire, UK) in 1% skim milk in PBST at room temperature
for 45 min. The membranes were washed four times with PBST. The band of amyloid fibrils
was detected by an ECL Western Blotting Detection System (GE Healthcare), and bands in
Western blot analysis were visualized by an LAS 4000 mini (Fujifilm, Tokyo, Japan).
After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB). Another set
of electrophoresed gels was transferred onto PVDF membranes (Millipore, Billerica, MA,
USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) supplemented with
0.1% Tween 20 (PBST). The membranes were incubated with anti-mouse SAA1 goat antibody
(1:5,000) (AF2948, R&D Systems, Minneapolis, MN, USA), anti-bovine SAA1 monoclonal
antibody, 25BF12 (1:1,600) [29 (link)], which was purified
from the culture supernatant of hybridoma cells by protein A-sepharose (GE Healthcare), or
anti-chicken SAA1 mouse antibody (1:1,600) [22 (link)] in
1% skim milk in PBST at room temperature for 1 h. Membranes were washed three times with
PBST and incubated with anti-goat IgG donkey antibody F (ab’)2 conjugated with
horseradish peroxidase (HRP) (1:1,000) (SC3851, Santa Cruz Biotechnology, Dallas, TX,
USA), anti-mouse IgG F (ab’)2 conjugated with HRP (1:1,600)
(NA9310V, GE Healthcare, Buckinghamshire, UK) in 1% skim milk in PBST at room temperature
for 45 min. The membranes were washed four times with PBST. The band of amyloid fibrils
was detected by an ECL Western Blotting Detection System (GE Healthcare), and bands in
Western blot analysis were visualized by an LAS 4000 mini (Fujifilm, Tokyo, Japan).
6
Western Blot Analysis of Smad Signaling
7
Western Blot Analysis of Key Proteins
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For western blotting analysis, cells were harvested, rinsed with PBS and lysed with ice-cold lysis buffer (50 mM TrisHCl pH = 7.4, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM sodium fluoride, protease, and phosphatase inhibitors). Western blotting was performed according to standard procedures. Membranes were incubated overnight with the following primary antibodies: anti-IGF2BP3 (RN009P, dilution 1:20000, MBL International, Woburn, MA, USA), anti-CXCR4 (ab124824, dilution 1:1000, Abcam), anti-CD164 (AF5790, dilution 1:1000, R&D Systems, Minneapolis, MN, USA), anti-HIF-1α (sc-10790, dilution 1:2000, Santa Cruz Biotechnology), and anti-GAPDH (sc-25778, dilution 1:10000, Santa Cruz Biotechnology). The following secondary antibodies were used: anti-rabbit (NA934) and anti-mouse (NA9310V, GE Healthcare, Little Chalfont, UK) or anti-sheep (HAF016, R&D Systems) antibodies conjugated to horseradish peroxidase.
8
Exosomal Protein Extraction and Analysis
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Total proteins were extracted from purified exosomes using RIPA buffer, sonication and vortexing. The exosomal proteins (10 μg) were run on a Mini-Protean TGX precast gel (Any kD, Bio-Rad Laboratories, Hercules, CA, USA) and blotted to Trans-Blot Mini PVDF membranes using the Trans-Blot Turbo™ Transfer system (Bio-Rad). Blots were incubated with blocking buffer (5% non-fat milk/PBST) overnight at 4°C. OVA was detected using an anti-OVA antibody (1:1,000; 0220-1682, AbD Serotec, Kidlington, UK), together with a donkey-anti-rabbit secondary antibody (1:10,000; NA9340, GE Healthcare, Little Chalfont, UK). Alix was detected using an anti-Alix antibody (1:1000; 2171 (3A9), Cell Signaling technology, Danvers MA, US), together with a sheep-anti-mouse secondary antibody (1:2000; NA9310V, GE Healthcare, Little Chalfont, UK) and visualized by enhanced chemiluminescence (GE Healthcare), the ChemiDoc™ MP Imaging System and Image Lab™ software version 4.1 (both from Bio-Rad).
9
Western Blot and Intracellular Staining
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Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 4–12% Criterion gels (BioRad, Hercules, California) with MOPS running buffer and transferred to cellulose membranes (GE Healthcare, Little Chalfont, United Kingdom) with the Criterion Blotter system (BioRad). The following antibodies were used: an anti-STrEP-Tag (#34850, Qiagen, Hilden, Germany), anti-LGP2 (NBP1-85348, Novus, Littleton, Colorado), anti-MDA5 (#5321, Cell Signaling, Danvers, Massachusetts and AT113, EnzoLifescience, New York, NY), anti-RIG-I (D14G6, Cell Signaling) or monoclonal anti-β-actin antibody (A5441, Sigma), IRF3 (#11904, Cell Signaling) or phosphor-IRF3 (ab76493, Abcam, Cambridge, UK,). HRP-coupled anti-mouse (NA9310V, GE Healthcare) or anti-rabbit (RPN4301, GE Healthcare) were used as secondary antibodies. Peroxidase activity was visualized with an ECL Plus Western Blotting Detection System (#RPN2132, GE Healthcare). MV intracellular staining was performed with mouse anti-N mAb (clone 25, [Giraudon and Wild, 1981 (link)]) and FITC coupled Goat Anti-mouse Ab (BD Biosciences, Franklin Lakes, New Jersey). For CHIKV, intracellular staining was performed with either FITC-conjugated anti-CHIK.E2 mAB 3E4 (Bréhin et al., 2008 (link)) or anti-dsRNA mAb (J2-1201, Scicons, Szirák, Hungary) followed by anti-mouse-APC antibody (A865, Invitrogen).
10
Western Blot Analysis of Mfn2 Protein
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Left ventricular tissue was homogenized in ice-cold lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% triton X-100, 50 mM NaF, 5 mM Na4P2O7, 1 mM Na3VO4, 1 mM DTT, protease inhibitor cocktail), and protein content was determined using the BCA assay. About 30 μg of protein was separated by 10% SDS-PAGE and transferred onto 0.2 μm PVDF membrane and blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBS-T). Blocked membranes were exposed to primary antibodies toward total Mfn2 (Abcam mouse monoclonal anti-Mfn2, ab56889, 1:2000 dilution) and GAPDH (Cell Signaling antibodies, 36,835, 1:2000 dilution). Sheep anti-mouse immunoglobulin G (IgG) antibodies (GE Healthcare NA9310V) in 1:4000 dilution were used as secondary antibodies. Protein bands were visualized by enhanced chemiluminescence (GE Healthcare Life Sciences). Mfn2 bands were normalized to GAPDH bands using the software ScanPack 3.0.
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