Fifteen 5 days post-fertilization (dpf) zebrafish larvae were lysed in 35 uL 4 × Laemmli buffer containing 0.4 mM DTT for 10 min at 96 °C with regular vortexing. Cell lysates were made in RIPA buffer and diluted in 4 × Laemmli buffer containing 5% β-mercaptoethanol. Equal amounts of protein from cells or zebrafish lysates were loaded on a 4–15% Criterion TGX Stain-Free Protein Gel and separated at 150 volt for 60 min in Tris–glycine-SDS running buffer. Proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare Life Sciences) for 10 min at 2.5A and 25 V. Membranes were blocked for 1 h in 0.1% PBS–Tween containing 5% low-fat milk powder (Elk, Campina). Incubation with the first antibody was performed overnight in blocking buffer at 4 °C. The following day, membranes were incubated with the secondary antibody for 1 h at room temperature (RT). Proteins were revealed with a fluorescent-based approach on the Odyssey Infrared Imager (LI-COR Biosciences).
Amersham protran 0.45 nc
Manufactured by GE Healthcare
Sourced in United States
The Amersham Protran 0.45 NC is a nitrocellulose membrane designed for use in Western blotting and other protein-based applications. It is made of nitrocellulose and has a pore size of 0.45 micrometers.
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Lab products found in correlation
Ecl reagent, by GE Healthcare (3 mentions)
El7 e1, by Abcam (2 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Ab109497, by Abcam (1 mentions)
Ab186321, by Abcam (1 mentions)
Beta 1 tubulin antibody clone e7, by Developmental Studies Hybridoma Bank (1 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Promega (1 mentions)
β actin, by Merck Group (1 mentions)
Anti ha antibody 3f10, by Roche (1 mentions)
Anti rad53 antibody el7 e1, by Abcam (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Twist1, by Cell Signaling Technology (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Pkmyt1, by Cell Signaling Technology (1 mentions)
Multiskan mk3, by Thermo Fisher Scientific (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
8 protocols using amersham protran 0.45 nc
1
Western Blot Profiling of Zebrafish
2
Western Blot Analysis of Rad53 Phosphorylation
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Aliquots of 5 × 107 cells were harvested by centrifugation. The pellets were lysed in 0.2 M NaOH for 10 min on ice, and proteins were precipitated by the addition of 50 µL of 50% trichloroacetic acid. The samples were centrifuged at 16,100g for 10 min at 4°C, and the pellets were resuspended in 4× Laemmli buffer and heated for 5 min at 95°C. Samples were separated in a denaturing 7.5% 37.5:1 polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare). The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (Abcam, EL7.E1) that recognizes both the unphosphorylated and phosphorylated forms of Rad53. Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare).
3
Western Blot Analysis of TSPO and VDAC1
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Whole cell protein samples were sonicated and boiled in RIPA buffer, and total protein was quantified using a micro-BCA colorimetric assay (Pierce, Thermo Fischer Scientific, Darmstadt, Germany). Protein samples were separated by SDS-polyacrylamide gel electrophoresis on 15% gels and subsequently transferred onto a nitrocellulose membrane (Amersham™ Protran 0.45 NC, GE Healthcare Life Sciences, Chicago, IL, USA). TSPO and VDAC1 proteins were detected using rabbit anti-TSPO (ab109497) and mouse anti-VDAC1 (ab186321) antibodies, both from Abcam, Cambridge, UK, respectively. Beta-1 tubulin antibody (clone E7, Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used as a loading control. VDAC1 band densities were quantified using ImageJ2 Version 2.9.2. [30 (link)] and normalized to beta-1 tubulin.
4
Western Blot Analysis of Protein Expression
5
Rad53 and Cdc5 Protein Analysis
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Aliquots of 5 × 107 cells were harvested by centrifugation. The pellets were lysed in 0.2 M NaOH on ice for 10 min and proteins were precipitated by the addition of 50 µl of 50% trichloroacetic acid. The samples were centrifuged at 16,100 × g for 10 min at 4°C and the pellets were resuspended in 4× Laemmli buffer and heated for 5 min at 95°C. Samples were separated in a denaturing 7.5% 37.5:1 polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare). The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 antibody (EL7.E1; Abcam), which recognizes both the unphosphorylated and phosphorylated forms of Rad53, anti-Cdc5 antibodies (11H12 and 4F10; Medimabs), anti-HA antibody (3F10; Roche), and anti-Pgk1 antibody (22C5D8; Abcam). Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare). All western blots were independently replicated in the laboratory.
6
Western Blot Analysis of EMT and PI3K/AKT Pathway
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All the protein was collected by using RIPA lysis buffer (Beyotime Biotechnology, P0013, Shanghai, China). Protein was quantified using Coomassie blue staining in microplate spectrophotometer (Thermo Scientific, Multiskan MK3, Waltham, MA, USA) at 594 nm. Identical amount of protein was separated into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then the gels were transferred onto nitrocellulose membrane (GE Healthcare Life Sciences, Amersham™ Protran™ 0.45 NC, Chicago, IL, USA) after electrophoresis. Membranes were blocked by 5% skim milk for 2 hrs. Then, the membranes were incubated with primary antibodies (E-cadherin (ECAD), N-cadherin (NCAD), Vimentin (VIM), and β-actin were purchased from Proteintech group, Wuhan, China; PKMYT1, AKT, mTOR, S6, Phospho-AKT, Phospho-mTOR, Phospho-S6, and Twist1 were purchased from Cell Signaling Technology, Danvers, MA, USA) in 5% bovine serum albumin overnight at 4°C. After rewarming to room temperature for 1 hr, the membranes were incubated with secondary antibody for 1 hrat 37°C. We used enhanced chemiluminescence (Beyotime Biotechnology, Shanghai, China) to detect the blots with ChemiDoc MP (Bio-Rad Laboratories, ChemiDoc MP, Hercules, CA, USA) and the images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Western Blotting of NS Proteins
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For Western blotting, the cells in a 6-well plate well were lysed and denatured in 150 μL of 6× Laemmli buffer by heating to 95°C for 5 minutes, and loaded onto an 12% polyacrylamide-SDS gel. After resolution by SDS-PAGE, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, with the exception of NS7, which was transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare Life Science). NS proteins were detected using NS-protein specific polyclonal rabbit antibodies described in IF section in a 1:1000 dilution. β-actin was detected by monoclonal mouse antibody (A5441), Sigma-Aldrich. Primary antibodies were detected using αrabbit/ αmouse horseradish peroxidase (HRP)-coupled secondary antibodies (Sigma-Aldrich) and imaging was done with the ChemoCam 6.0 ECL system (INTAS Science Imaging, Goettingen, Germany).
8
Rad53 Phosphorylation Analysis by Western Blot
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Aliquots of 5 × 10 7 cells were harvested by centrifugation. The pellets were lysed in 0.2 M NaOH on ice for 10 min and proteins were precipitated by the addition of 50 µL of 50% trichloroacetic acid. The samples were centrifuged at 16,100 g for 10 min at 4°C and the pellets were resuspended in 4× Laemmli buffer and heated for 5 min at 95°C. Samples were separated in a denaturing 7.5% 37.5:1 polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare).
The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (EL7.E1, Abcam), which recognizes both the unphosphorylated and phosphorylated forms of Rad53, anti-Cdc5 (11H12 and 4F10, Medimabs), anti-HA primary antibody (3F10, Roche), and anti-Pgk1 primary antibody (22C5D8, Abcam). Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare). All western blots were independently replicated in the laboratory.
The membranes were stained with Ponceau Red and immunoblotted with anti-Rad53 primary antibody (EL7.E1, Abcam), which recognizes both the unphosphorylated and phosphorylated forms of Rad53, anti-Cdc5 (11H12 and 4F10, Medimabs), anti-HA primary antibody (3F10, Roche), and anti-Pgk1 primary antibody (22C5D8, Abcam). Blots were then incubated with a horseradish peroxidase-coupled secondary antibody, and the signal was detected using ECL reagent (Amersham, GE Healthcare). All western blots were independently replicated in the laboratory.
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