Cochlear explants were lysed with RIPA buffer containing a protein phosphatase inhibitor cocktail (Roche Applied Science, Germany) and centrifuged for 10 min at 12000 rpm. Protein concentrations of supernatants were determined using a BCA assay kit (Beyotime Institute of Biotechnology, Shanghai, China) and then equal amounts of protein were denatured by heating for 10 min at 90°C after adding 5×SDS-PAGE loading buffer. Proteins were then separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked in TTBS containing 5% BSA for 1 h and then incubated with different primary antibodies (AKT, 1:5000, phospho-AKT, 1:500, GSK3β,1:1000, phospho-GSK3β,1:2000, NF-κB p65 (Ser536), 1:1000; phospho-p65, 1:500) in TTBS containing 3% BSA overnight at 4°C. Membranes were then incubated with an HRP-labeled secondary antibody at room temperature for 2 h, and then protein bands were visualize using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA). Images were obtained using a Dolphin‑C image system (Wealtec Corp., Sparks, NV, USA). Immunoblots were quantified using the gray values for each protein band using Image J software. Relative protein levels were calculated using β-actin as an internal standard. All experiments were performed in triplicate.
Protein phosphatase inhibitor cocktail
Manufactured by Roche
Sourced in Germany
Protein phosphatase inhibitor cocktail is a laboratory reagent used to inhibit the activity of protein phosphatases. Protein phosphatases are enzymes that remove phosphate groups from proteins, and their inhibition can help preserve the phosphorylation state of proteins in biological samples. This product is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (7 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (2 mentions)
Protein a sepharose p3391, by Merck Group (2 mentions)
Prmt5 mep50 active complex srp0145, by Merck Group (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura extended duration substrate kit, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Mouse anti smn antibody, by BD (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pt389 p70s6k, by Cell Signaling Technology (1 mentions)
Rictor, by Cell Signaling Technology (1 mentions)
Raptor, by Cell Signaling Technology (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Anti mecp2, by Abcam (1 mentions)
Protran ba 85 nitrocellulose membranes, by Cytiva (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab32360, by Abcam (1 mentions)
Ab9572, by Abcam (1 mentions)
10 protocols using protein phosphatase inhibitor cocktail
1
Western Blot Analysis of Cochlear Protein
2
SARS-CoV-2 Protein Detection by Immunoblotting
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Immunoblotting was performed using the following antibodies: mouse anti-Flag monoclonal antibody (F1804, Sigma Aldrich, 1:2000); rabbit anti-SARS-CoV-2 N antibody (1:2000, 9103, Prosci); rabbit anti-SARS-CoV-2 S antibody (1:1000, PA5-81795, Invitrogen); rabbit anti-TDRD3 antibody (1:1000, Bethyl Laboratory); mouse anti-SMN antibody (1:2000, 610646, BD Biosciences); rabbit anti-G3BP1 antibody (1:1000, 1F1, Rhône-Poulenc Rorer, a kind gift from Dr Imed Gallouzi at McGill University) (87 (link)); rabbit anti-ASYM26 (1:1000, 13-0011, EpiCypher). Immunofluorescence was performed with the following antibodies: mouse anti-Flag monoclonal antibody (F1804, Sigma Aldrich, 1:500); rabbit anti-G3BP1 antibody (1F1, 1:500). Alexa Fluor–conjugated goat anti-rabbit, goat anti-mouse secondary antibodies were from Invitrogen. Protease inhibitor cocktail and protein phosphatase inhibitor cocktail were from Roche. MS023 (SML1555), sodium arsenite (S7400), Protein A-Sepharose (P3391), and PRMT5:MEP50 active complex (SRP0145) were purchased from Sigma Aldrich. Protein coimmunoprecipitation was performed using ANTI-FLAG M2 Affinity Gel (A2220, Sigma Aldrich).
3
Immunoblotting and Immunoprecipitation Reagents
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Protease inhibitor cocktail (Complete mini EDTA-free protease inhibitors, Roche, 04693159001), protein phosphatase inhibitor cocktail (Phosphostop phosphatase inhibitors, Roche, 04906837001), anti-mTOR (T-2949) for immunoprecipitation and Histopaque-1077 were purchased from Sigma/Millipore. Anti-mTOR (SC-517464) for Western blotting, -pS2448mTOR (SC-293133), -pS2481mTOR (SC-293089), -Rictor (SC-271081), -Raptor (SC-81537), -p70S6Kα (SC-8418, SC-393967), -Akt1 (SC-5298, SC-55523), -pS473Akt (SC-514032), -FLNA (SC-58764, SC-17749, SC-271440), -IRβ (SC-09), and -PTEN (SC-7941, Cell Signaling #9559) were purchased from Santa Cruz Biotechnology. Anti-pS2152FLNA antibody (TA313881) was purchased from Origene Technologies. Anti-pT1135Rictor (#3806) and -pT389p70S6K (#9205) were purchased from Cell Signaling Technology. Insulin (human recombinant; #12585014), zinc solution (#12585014), covalently conjugated protein A/G-agarose beads (#20423), PageRuler™ Plus Prestained Protein Ladder, 10–250 kDa (#26619) and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34577) were purchased from ThermoFisher Scientific.
4
Western Blot Analysis of Hippocampal Proteins
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For western blot analysis, hippocampi of 8~12 weeks old mice were used. Mice were deeply anesthetized with isoflurane and decapitated; then, their hippocampi were removed and frozen immediately in liquid nitrogen and stored at -80℃ until lysis with a lysis buffer (1.6% SDS, 10 mM TrisCl; pH 6.8). Protease inhibitor cocktail (Roche 11873580001) and protein phosphatase inhibitor cocktail (Roche 04906845001) were also added to the lysis buffer. Hippocampal tissue was lysed in 400 µl lysis buffer with the QIAGEN TissueLyser LT (50 Hz, 3 min). For analysis of cleaved Notch1, mice were given a mild foot shock (0.5 mA, 2 sec) using the same protocol as for contextual fear conditioning. Fifteen micrograms of the protein samples were run by electrophoresis, and the blotted membranes were immunolabeled with antibodies against phospho-GluRepsilon2 (pGluN2B, Sigma-Aldrich, M2442), GluRepsilon2 (GluN2B, Neuromab, 75-097), Actin (Sigma-Aldrich, A2066), Tubulin (NeuroMab 75-330), cleaved Notch1 (CST, 2421) and Mib2 (Abnova H00142678-M01A). Mib1 antibody was gift from Dr. P. Gallagher.
5
Quantitative Western Blot Analysis of MeCP2 Phosphorylation
6
Western Blot Protein Analysis
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The cell lysates were prepared in RIPA lysis buffer supplemented with Protease Inhibitor Cocktail and Protein Phosphatase Inhibitor Cocktail (Roche, Upper Bavaria, Germany) on ice. The cell debris was discarded after refrigerated centrifugation, and the supernatant was collected for quantification using the BCA Protein Assay Kit (ThermoFisher, USA). The equal amount of protein sample was resolved by SDS‐PAGE gel and transferred to PVDF membrane on ice. The PVDF membrane was blocked with 5% skim milk in TBST buffer at room temperature for 1 hour and hybridized with indicated primary (anti‐p50, ab32360, Abcam, Cambridge, MA, USA 1:1000; anti‐IGF‐1, ab9572, Abcam, 1:1000; anti‐IL‐10, ab34843, Abcam, 1:1000; anti‐GAPDH, ab9485, Abcam, 1:1000) antibodies at 4°C overnight. After rigorous wash with TBST for 5 minutes by 6 times, the PVDF membrane was incubated with horseradish‐conjugated secondary antibodies (anti‐mouse, ab97046, Abcam, 1:5000; anti‐rabbit, ab6721, Abcam, 1:5000) at room temperature for 1 hour. The free antibodies were completely washed off with TBST for 5 minutes by 6 times, and the protein bands were visualized using the enhanced chemiluminescence kit (ELC, Millipore, Billerica, MA, USA) and images were acquired with ChemiDoc Touch Imaging System (Bio‐Rad, Hercules, CA, USA).
7
Protein Extraction and Western Blot Analysis
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Cells were washed with ice-cold Dulbecco’s phosphate-buffered saline (DPBS; Hyclone, Logan, Utah), and then lysed for 30 min on ice using RIPA buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid, 1% Triton X-100, protease inhibitor cocktail tablets (Roche, Penzberg, Germany), protein phosphatase inhibitor cocktail (Roche), and 1 mM PMSF. Protein concentration was determined using a BCA assay kit (Pierce, Rockford, IL). Equal amounts of protein (20 μg) were dissolved in sample buffer containing 50 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, 0.01% bromophenol blue, and 10% glycerol and separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. This was followed by transfer onto polyvinylidene difluoride membranes. Blots were probed with appropriate antibodies (1:1,000): anti-ICAM1, anti-β-actin, anti-G9a, anti-KDM4B, or anti-VCAM1. Anti-rabbit and anti-mouse horseradish peroxidase-conjugated IgG (1:5,000) were used as secondary antibodies; the blots were subsequently developed using enhanced chemiluminescence (ECL) reagents (Advansta, Menlo Park, CA).
8
Antibody-based Investigation of DNA Repair Factors
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Mouse anti-DDX5 (A-5, sc-166167) monoclonal and rabbit anti-RAD51 (H-92, sc-8349) and anti-Ku80 (H-300, sc-9034) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-53BP1 (NB100-304) was from Novus Biologicals (Littleton, CO). Mouse anti-γH2AX (05-636), anti-p68 (DDX5) (clone204, 05-850) monoclonal antibodies, and rabbit anti-BRCA1 antibody (07-434) were obtained from Millipore (Billerica, MA). Anti-cyclin A (BD611268) monoclonal antibody was from BD Biosciences (San Jose, CA). Anti-BrdU antibody (RPN202) was from Cytiva. V5 mouse mAb antibody was from Life Technologies (#R96025). S9.6 antibody was purified, in house, from the hybridoma (ATCC® HB-8730). Propidium iodide and the Alexa Fluor-conjugated goat anti-rabbit antibodies and anti-mouse antibodies were from Invitrogen (Carlsbad, CA). Escherichia coli RNase H was purchased from New England Biolabs. Bromo-2′-deoxyuridine (BrdU), 4-hydroxytamoxifen (4-OHT), protein A Sepharose, mouse anti-Flag, and α-tubulin monoclonal antibodies were from Millipore-Sigma (St Louis, MO). For DRIP, Pierce™ Protein A/G UltraLink™ Resin was purchased from Thermo Fisher (#53133). Shield 1 was purchased from Clontech Laboratories (Mountain View, CA). Protease inhibitor cocktail and protein phosphatase inhibitor cocktail were purchased from Roche (Mississauga, ON, Canada).
9
Tissue Lysis and Protein Extraction
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To prepare a whole lysate solution, the collected tissues were washed twice with ice-cold PBS and lysed in a lysis buffer (Beyotime, Shanghai, China) that contained 1 mM of phenylmethylsulfonyl fluoride (PMSF) and a protein phosphatase inhibitor cocktail (Roche, Basel, Switzerland) for 10 min on ice. The homogenates were centrifuged at 12,000 rpm at 4 °C for 10 min. Total protein was extracted from the scraped colonic mucosa of the experimental mice using a NE-PER nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA), following the manufacturer's recommendations. Protein concentration was determined using a Bio-Rad DC Protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following the manufacturer's instructions.
10
SARS-CoV-2 Immunoblotting and Immunofluorescence
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Immunoblotting was performed using the following antibodies: mouse anti-Flag monoclonal antibody (F1804, Sigma Aldrich, 1:2000); rabbit anti-SARS-CoV-2 N antibody (9103, Prosci, 1:2000); rabbit anti-SARS-CoV-2 S antibody (PA5-81795, Invitrogen, 1:1000); rabbit anti-TDRD3 antibody (Bethyl Laboratory, 1:1000); mouse anti-SMN antibody (610646, BD Biosciences, 1:2000); rabbit anti-G3BP1 antibody (1F1, Rhône-Poulenc Rorer, 1:1000, a kind gift from Dr. Imed Gallouzi at McGill University) 86 ; rabbit anti-ASYM26 (13-0011, Epicypher, 1:1000). Immunofluorescence were performed with the following antibodies: mouse anti-Flag monoclonal antibody (F1804, Sigma Aldrich, 1:500); rabbit anti-G3BP1 antibody (1F1, 1:500).
Alexa Fluor-conjugated goat anti-rabbit, goat anti-mouse secondary antibodies were from Invitrogen. Protease inhibitor cocktail and protein phosphatase inhibitor cocktail were from Roche. MS023 (SML1555), sodium arsenite (S7400), Protein A-Sepharose (P3391) and PRMT5:MEP50 active complex (SRP0145) were purchased from Sigma Aldrich. Protein coimmunoprecipitation was performed using ANTI-FLAG® M2 Affinity Gel (A2220, Sigma Aldrich).
Alexa Fluor-conjugated goat anti-rabbit, goat anti-mouse secondary antibodies were from Invitrogen. Protease inhibitor cocktail and protein phosphatase inhibitor cocktail were from Roche. MS023 (SML1555), sodium arsenite (S7400), Protein A-Sepharose (P3391) and PRMT5:MEP50 active complex (SRP0145) were purchased from Sigma Aldrich. Protein coimmunoprecipitation was performed using ANTI-FLAG® M2 Affinity Gel (A2220, Sigma Aldrich).
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