Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galectin‐1 antibody (1:1000, R&D systems), rabbit antibodies against ATF2 (1:1000), phosphorylated ATF2 (1:1000), c‐Fos, phosphorylated c‐Fos (1:1000), c‐Jun (1:1000), phosphorylated c‐Jun (1:1000), AKT (also known as protein kinase B) (1:1000), phosphorylated AKT (1:1000), ERK1/2 (1:1000), phosphorylated ERK1/2 (1:1000, Cell Signaling Technology), DUSP1 (1:1000, Millipore) and β‐actin (1:4000, Medical & Biological Laboratories). Horseradish peroxidase‐conjugated anti‐goat and anti‐rabbit IgGs (1:4000, Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were obtained by enhanced chemoluminescence (Perkin Elmer).
Phosphatase inhibitor cocktail
Manufactured by Fujifilm
Sourced in Japan
The Phosphatase Inhibitor Cocktail is a solution designed to inhibit the activity of phosphatases, enzymes that remove phosphate groups from proteins. This product can be used to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Fujifilm (5 mentions)
Protease inhibitor cocktail, by Promega (3 mentions)
Bca reagent, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemoluminescence, by PerkinElmer (1 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Dusp1, by Merck Group (1 mentions)
β actin, by Medical & Biological Laboratories (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk antibody, by Cell Signaling Technology (1 mentions)
Anti phospho 4e bp1 antibody, by Cell Signaling Technology (1 mentions)
α tubulin antibody, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Rabbit anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Rabbit anti phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Rabbit anti erk1 2, by Cell Signaling Technology (1 mentions)
13 protocols using phosphatase inhibitor cocktail
1
Western Blot Analysis of Signaling Proteins
2
Phosphorylation Signaling in PG Cells
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Dissected PGs were pre-cultured in Grace's culture medium for 30 min, then cultured with or without PDF or PTTH for 15 min. The cultured PGs were sonicated in 50 mM Tris-HCl (pH 6.8) with protease inhibitor cocktail (Complete, Roche) and phosphatase inhibitor cocktail (Wako), lysed in 1x SDS sample buffer excluding 2-mercaptoethanol (2ME) and bromophenol blue (BPB), and the protein concentration was measured using the BCA protein assay kit (Pierce). After adding 2ME and BPB, 10 µg of protein was loaded in each well and separated on a 12% SDS-PAGE gel. Anti-phospho-ERK antibody, anti-phospho-4E-BP1 antibody, anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody, and anti-mouse IgG HRP-linked antibody were purchased from Cell Signaling Technology (cat #9101, #9459, #7074, #7076), and the α-tubulin antibody was purchased from Sigma-Aldrich (cat #T9026). Immunostar LD (Wako) was used for signal detection.
3
Immunoprecipitation and Immunoblot Analysis
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Cells were homogenized in lysis buffer (1% NP‐40 in PBS) with a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After sonicated cell extracts were preabsorbed with Dynabeads Protein G (Thermo Fisher Scientific), the cell extracts were incubated with antibodies and Dynabeads Protein G overnight at 4°C with gentle mixing. The beads were washed with the lysis buffer and suspended in SDS sample buffer. The eluted proteins were analysed by immunoblot analysis.
4
Medaka Cdk9 Coimmunoprecipitation Protocol
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Preovulatory follicles isolated from ovaries 7 h before ovulation were sonicated for a few seconds in 50 mM Tris-HCl (pH 8.0) containing a 1 × protease inhibitor cocktail (Wako) and 1× phosphatase inhibitor cocktail (Wako), and then centrifuged at 13,000× g for 10 min. The resulting supernatants were used for coimmunoprecipitation analysis. The samples were treated with Protein G-Sepharose (GE Healthcare) that had been previously coupled with anti-medaka Cdk9 antibody. After incubation at 4 °C for 16 h, they were washed four times using 50 mM Tris-HCl (pH 8.0). The precipitant materials were boiled in 1× SDS sample buffer for 10 min and used for western blot analyses.
5
Immunoblotting Analysis of Protein Signaling
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Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galecint‐1 (1:1000, R&D systems), mouse anti‐TSC22D3 (1:500), mouse anti‐ubiquitin (1:500, Santa Cruz Biotechnology), rabbit anti‐HIF‐1α (1:1000), rabbit anti‐phosphorylated AKT (1:2000), rabbit anti‐AKT (1:2000), rabbit anti‐phosphorylated ERK1/2 (1:2000), rabbit anti‐ERK1/2 (1:2000, Cell signaling technology) and rabbit anti‐β‐actin (1:4000, Medical & Biological Laboratories) antibodies. Horseradish peroxidase‐conjugated anti‐goat, anti‐mouse and anti‐rabbit IgGs (Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
6
Quantification of Amyloid-Beta Levels
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Aβ levels were measured as described in a previous report [26 (link)]. Briefly, cortical tissues of the left hemisphere (Tg2576/UCP1+/+: six females, Tg2576/UCP1−/−: two males and four females) were frozen in liquid nitrogen and stored at −80 °C until Aβ ELISA analysis. The cortical tissues were homogenized in Tris-buffered saline (TBS) containing a phosphatase inhibitor cocktail and a protease inhibitor cocktail (Wako Pure Chemical Industries, Osaka, Japan). The homogenates were centrifuged at 100,000 rpm for 20 min at 4 °C. The resulting supernatants were used to determine the soluble Aβ levels. For insoluble Aβ determination, the pellets were dissolved in 6 M guanidine chloride, following sonication, incubation for 1 h at RT, and centrifugation at 100,000 rpm for 20 min at 4 °C. Human/Rat β Amyloid (Aβ40) ELISA kits (#294-64071, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and Human/Rat β Amyloid (Aβ42) ELISA kits (#290-62601, FUJIFILM Wako Pure Chemical Corporation) were used according to the manufacturer’s instructions. The obtained Aβ levels were normalized to the brain tissue weight.
7
Quantification of Soluble and Insoluble Aβ
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Inhalation of CO2 was used to kill the mice, which were then perfused with 0.1 M PBS. The brains of all mouse samples were removed and the cortical tissues of the left hemisphere were snap-frozen in liquid nitrogen and stored at − 80 °C until biochemical analysis, as described previously49 (link). The cortical tissues were homogenized in 19 volumes of Tris-buffered saline (TBS; pH 7.6, 10 mM Tris and 150 mM NaCl) containing a phosphatase inhibitor cocktail (Wako Pure Chemical Industries, Osaka, Japan) and a protease inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged at 100,000 rpm for 20 min at 4 °C. The supernatants were used for soluble Aβ measurements. After washing the pellets with TBS, the pellets were dissolved in 10 volumes of 6 M guanidine chloride, sonicated, and incubated for 1 h at RT. They were then centrifuged at 100,000 rpm for 20 min at 4 °C, and the resulting supernatants were used for insoluble Aβ measurements. Aβ40 and Aβ42 levels were assessed using ELISA kits (Wako) in accordance with the manufacturer’s instruction. The Aβ levels were normalized to the weight of the tissue.
8
Immunoblot Analysis of Protein Expression
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For immunoblot analysis, total proteins were extracted from the control fibroblasts, ciBAs, and Capsaicin-treated ciBAs with RIPA buffer (FUJIFILM Wako) including phosphatase inhibitor cocktail (FUJIFILM Wako) and protease inhibitor cocktail (FUJIFILM Wako). The extracted proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 10% gel concentration and transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, MA, USA). The membranes were blocked with 3% skim milk followed by incubation with antibodies against UCP1 (MAB6158, R&D Systems, MN, USA), COX4 (#4850, Cell Signalling Technology, MA, USA), VDAC1 (ab14734, Abcam, Cambridge, UK), or β-Actin (A5316, Sigma-Aldrich) at 4 ˚C overnight. The membranes were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). The intensity of each band was quantified by densitometry using ImageJ software (National Institutes of Health). The experiments were performed independently at least twice.
9
Hippocampal Protein Extraction and Western Blotting
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N 2 -Flushed hippocampal tissues were homogenized with a lysis buffer composed of 1 mM EDTA, 1% SDS, 1× complete protease inhibitor cocktail (Roche Diagnostics, Schweiz), 1× phosphatase inhibitor cocktail (Fujifilm Wako Pure Chemical Corporation), and 20 mM Tris-HCl ( pH 7.4). After incubation on ice for 30 min, the supernatant was collected by centrifugation at 13 000g for 15 min at 4 °C. Proteins were separated on 10% Tris-HCl denaturing gels by SDS-PAGE and transferred onto polyvinyl difluoride membranes (Immobilon-P, Merck Millipore, Burlington, MA, USA), as described previously. 36, [41] [42] [43] The membranes were blocked with 5% skimmed milk (Fujifilm Wako Pure Chemical Corporation),
and incubated with primary antibodies (1 : 1000) overnight at 4 °C, followed by washing five times with Tris-buffered saline.
The HRP-conjugated secondary antibodies were detected using Amarsham ECL Prime Western detection reagent (GE Healthcare) and visualized using an Image Quant LAS-4000 biomolecular imager (FUJI FILM, Tokyo, Japan). The protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
and incubated with primary antibodies (1 : 1000) overnight at 4 °C, followed by washing five times with Tris-buffered saline.
The HRP-conjugated secondary antibodies were detected using Amarsham ECL Prime Western detection reagent (GE Healthcare) and visualized using an Image Quant LAS-4000 biomolecular imager (FUJI FILM, Tokyo, Japan). The protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
10
Recombinant Protein Binding Assay
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To prevent recombinant protein degradation and aggregation, the pull-down assay was performed with freshly prepared recombinant proteins. His6tagged ezrin proteins were mixed with glutathione beads bearing each GST-fused protein prepared as described above in binding buffer [20 mM Tris-Cl ( pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100 and 150 mM imidazole] and incubated for 2 h at 4°C. MDA-MB-231 cells stably expressing FLAG-tagged ezrin were lysed in lysis buffer [20 mM Tris-Cl ( pH 7.5), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100] containing protease inhibitor cocktail (Wako) and phosphatase inhibitor cocktail (Wako). Cell lysates were mixed with glutathione beads bearing GST fusion proteins prepared as described above in binding buffer [20 mM Tris-Cl ( pH 7.5), 150 mM NaCl, 10% glycerol and 1%Triton X-100] containing protease inhibitor cocktail and phosphatase inhibitor cocktail, and incubated for 2 h at 4°C. Beads were washed six times with binding buffer, and bound proteins were then eluted with SDS-PAGE sample buffer and subjected to western blot analysis. GST-fused proteins were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining.
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