For western blotting analysis, muscle samples were homogenized and analyzed as described previously (Takegaki et al., 2017 ). Briefly, muscle samples were homogenized in RIPA buffer (Thermo Fisher Scientific, Waltham, MA) containing HaltTM protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and were centrifuged at 10,000g for 10 min at 4°C. After determination of supernatant concentrations, samples were diluted in 3× Blue Loading Buffer (Cell Signaling Technology, Danvers, MA) and boiled at 95°C for 5 min. Equal amount of proteins (20 μg) were then subjected to 7.5%, 10%, or 12% TGX gel (BioRad, Hercules, CA) and subsequently transferred to Polyvinyliden difluoride (PVDF) membranes. Membranes were blocked in 5% skim milk in Tris‐buffered saline with Tween 20 (TBST) for 1 h at room temperature and subsequently incubated with the primary antibodies overnight at 4°C. Membranes were then incubated for 1 h with the appropriate secondary antibodies at room temperature and visualized using chemiluminescent reagents (ClarityTM Western ECL Substrate, BioRad). Bands were detected and quantified with ChemiDoc XRS (170‐8071, Bio‐Rad).
Blue loading buffer
Manufactured by Cell Signaling Technology
Sourced in United States
Blue Loading Buffer is a laboratory reagent used to prepare samples for gel electrophoresis. It contains a blue dye that allows visual monitoring of sample migration during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Tgx gel, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
X ray film, by Kodak (1 mentions)
Protease inhibitor, by Cell Signaling Technology (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Vardenafil, by Merck Group (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
20 protocols using blue loading buffer
1
Western Blotting Analysis of Muscle Samples
2
Western Blot Protein Analysis Protocol
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This assay was performed as described before 13 (link). Briefly, equal amounts of extracted proteins were prepared with RIPA buffer, protease inhibitors (Cell Signaling Technology), and a BCA assay kit (Thermo Fisher). The proteins were mixed with 3× blue loading buffer (#7722, Cell Signaling Technology) and denatured in a boiling water bath (100 °C) for 5 min and then separated in 12% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes. 5% milk was then used to block the blots for 1 h. Primary antibodies and horseradish peroxidase-conjugated secondary antibodies were each incubated for 1 h. The bound secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan). PBS-T was used to wash the PVDF membranes 3 times during each incubation.
3
Western Blot Analysis of Exosome Treatments
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NHKs were harvested at 24 or 48 h after Exo treatment and lysed in a radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). The detailed methods have been described previously [63 (link)]. The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were mixed with 3× blue loading buffer (Cell Signaling Technology, Danvers, MA, USA) and heated for 3 min at 95 °C. Samples were subjected to gel electrophoresis, electro-transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked with a 5% (w/v) bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (PBST) for 1 h at room temperature. The PVDF membranes were incubated with the primary antibodies (Table 2 ). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:3000; Millipore, Billerica, MA, USA) and HRP-conjugated goat anti-mouse IgG antibody (1:3000; Millipore). Images were obtained using a chemiluminescence imaging system (WSE-6100; ATTO, Tokyo, Japan) and the optical density of the bands was measured using the Image J software (Version 1.53, NIH, Bethesda, MD, USA). Protein expression was normalized to that of β-actin; the ratio of Exo-treated cells to DPBS-treated control cells was calculated (DPBS-treated control cells = 1.0).
4
Western Blot Protein Analysis
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Protein lysates were adjusted to the same concentration, mixed with 3× Blue Loading Buffer (Cell Signaling Technology), heated at 95°C for 5 minutes, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes by a wet tank transfer system. Membranes were blocked with 3% BSA in TBST buffer and probed with primary antibodies as indicated in Figure 1 D. Afterwards, membrane were incubated with HRP-conjugated secondary antibodies, developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific), and images acquired by Amersham Imager 600 (GE Healthcare).
5
High-Throughput Compound Screening for Drug Discovery
6
Protein Extraction from Prostate Tissues
7
Protein Extraction and Western Blot Analysis
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Cell lysates were harvested from cell monolayers using a lab‐made standard lysis buffer (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 2 mM EDTA, 10% [v/v] Glycerol, 1% [v/v] Triton X‐100) supplemented with a cocktail of protease inhibitors (Thermo Fisher Scientific), 1× blue loading buffer (Cell Signaling Technology, Danvers, MA, USA) and 1x DTT (Cell Signaling Technology). In some cases, to enhance the levels of CTFs, cell lysates were prepared by first detaching cells from the surface with TrypLE Express (ThermoFisher Scientific) for 1–2 min, washed once with PBS, before the addition of lysis buffer. Proteins in cell lysates were separated in precast 10% Tris–glycine gels (Thermo Fisher Scientific) by electrophoresis and subsequently transferred to PVDF membranes (Millipore, Billerica, MA, USA) by the Trans‐Blot Turbo system (Bio‐Rad, Hercules, CA, USA). Primary antibodies (Table S1 ) were incubated with the membranes at 4 °C overnight, which was followed by HRP‐conjugated secondary antibody (Table S1 ) incubation at room temperature for 1 h. Protein bands were developed with LumiGLO reagent (Cell Signaling Technology) according to the manufacturer's protocol and detected using a ChemiDoc Imaging System (Bio‐Rad). Protein content was analyzed by densitometric analysis using the NIH image j software (RRID:SCR_003070, 1.48v) and normalized to GAPDH.
8
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Cells were lysed in 1× blue loading buffer (#7722; Cell Signaling Technology, Inc.). Equal amounts of cell extracts were fractioned by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose membranes (400 mA for 1.5 hours; Bio-Rad). Membranes were blocked in 5% nonfat milk in TBST (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween 20), and then incubated with antibodies against various proteins: β-tubulin #2128 (Cell Signaling Technology, Inc.); GAPDH sc25778 (Santa Cruz Biotechnology), E-cadherin #3195 (Cell Sig-naling Technology, Inc.), N-cadherin #GTX112733, Twist1 #GTX121924, and Slug #GTX127310 (Genetex, South San Francisco, CA, USA). Horseradish peroxidase-labeled anti-rabbit antibody (#7074; Cell Signaling Technology, Inc.) was detected by chemiluminescence (Fisher Scientific, Pittsburgh, PA, USA). Signals were captured by a luminescent image analyzer (LAS4000; Fujifilm, Tokyo, Japan).
9
Protein Isolation and Detection Protocols
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Western blots: cells were lysed in Blue Loading Buffer (cat# 7722, Cell Signaling Technology), sonicated with a Misonix sonicator (Qsonica, LLC. Newtown, CT), boiled for 10min at 95°C before loading, and analyzed by SDS-PAGE immunoblot.
Co-immunoprecipitation: cells were lysed in Pierce IP Lysis Buffer (Thermo Fisher Scientific) and 1x Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were incubated with Anti-FLAG M2 Magnetic Beads (MilliporeSigma, Darmstadt, Germany) according to manufacturer instructions. Proteins were eluted using 3xFLAG peptide (MilliporeSigma, Darmstadt, Germany) and analyzed by SDS-PAGE immunoblot. FLAG-tagged proteins were visualized using Pierce ECL (Thermo Scientific) after treatment with an HRP-conjugated primary antibody directed against the FLAG epitope (Millipore Sigma). HRP-conjugated secondary antibodies against murine and rabbit antibodies to other targets (see below) plus ECL Prime reagent (Cytiva Amersham) were used to visualize all other proteins.
Co-immunoprecipitation: cells were lysed in Pierce IP Lysis Buffer (Thermo Fisher Scientific) and 1x Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were incubated with Anti-FLAG M2 Magnetic Beads (MilliporeSigma, Darmstadt, Germany) according to manufacturer instructions. Proteins were eluted using 3xFLAG peptide (MilliporeSigma, Darmstadt, Germany) and analyzed by SDS-PAGE immunoblot. FLAG-tagged proteins were visualized using Pierce ECL (Thermo Scientific) after treatment with an HRP-conjugated primary antibody directed against the FLAG epitope (Millipore Sigma). HRP-conjugated secondary antibodies against murine and rabbit antibodies to other targets (see below) plus ECL Prime reagent (Cytiva Amersham) were used to visualize all other proteins.
10
MDM-T Lymphocyte Protein Analysis
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MDM or MDM-T lymphocyte co-cultures were lysed in Blue Loading Buffer (Cell Signaling), sonicated with a Misonix sonicator (Qsonica, LLC.) and clarified by centrifugation at 13000 RPM. Lysates were analyzed by SDS-PAGE immunoblot and protein levels were quantified using Adobe Photoshop as described [11 ,49 (link)].
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