For YB-1 phosphorylation studies, cells were lysed in RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 7.6) supplemented with 1× protease/phosphatase inhibitor cocktail (Cell Signaling). For co-immunoprecipitation studies, cells were lysed in NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-Cl pH 8.0) supplemented with 1× protease/phosphatase inhibitor cocktail (Cell Signaling). For RNase treatment, 3.33 μg of RNase A (Thermo-Fisher) was added to 1000 μg of protein lysate and incubated at 25°C for 10 minutes. Lysates were incubated on ice for 30 minutes and then cleared by spinning at 10,000 RPM for 10 minutes at 4°C. Supernatants were incubated with GFP-Trap agarose beads (Chromotek) for 2 hours at 4°C or FLAG agarose beads at 4°C overnight. Beads were washed three times with RIPA or NP-40 lysis buffer afterward for YB-1 phosphorylation and co-immunoprecipitation studies respectively. Bound proteins were eluted with 4× Laemmli Sample Buffer (BioRad) and visualized by SDS-PAGE on nitrocellulose membranes followed by immunoblot analysis. A portion of each protein lysate input was run concurrently to measure total expression for each protein.
Protease phosphatase inhibitor cocktail
Manufactured by Cell Signaling Technology
Sourced in United States, China, Germany, Japan
Protease/phosphatase inhibitor cocktail is a lab equipment product that contains a mixture of chemical inhibitors designed to prevent the degradation of proteins and the dephosphorylation of phosphoproteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (30 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (25 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (20 mentions)
Bca kit, by Thermo Fisher Scientific (15 mentions)
Cell lysis buffer, by Cell Signaling Technology (14 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (14 mentions)
Ripa buffer, by Cell Signaling Technology (12 mentions)
β actin, by Cell Signaling Technology (11 mentions)
Nitrocellulose membrane, by Bio-Rad (10 mentions)
Ripa buffer, by Merck Group (9 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Bis tris gel, by Thermo Fisher Scientific (8 mentions)
Laemmli sample buffer, by Bio-Rad (7 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (6 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Anti β actin, by Cell Signaling Technology (6 mentions)
Chemidoc mp system, by Bio-Rad (6 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (5 mentions)
304 protocols using protease phosphatase inhibitor cocktail
1
YB-1 Phosphorylation and Co-Immunoprecipitation Assay
2
YB-1 Phosphorylation and Co-Immunoprecipitation Assay
3
Isolation and Lysis of Cells and Extracellular Vesicles
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PC9 or LK2 cells were washed with PBS twice and then incubated with Accutase for 5 min at 37 °C and re-suspended in the complete media (RPMI 1640 medium, supplemented with GlutaMAX™ (Gibco®) and 10% exosome-depleted FB). Cell number was quantified using Vi-CELL™ XR Cell Viability Analyzer and cells were washed with PBS twice and spun down for 5 min at 322×g. Cell pellets were kept at −80 °C. Cells were lysed in RIPA Lysis and Extraction Buffer (Pierce, #89900) supplemented with Protease/Phosphatase Inhibitor cocktail (Cell Signalling Technology, 5872 S, 1:100) at the ratio 5*106 cells per 300 µl of lysis buffer. Next, cell lysates were ultrasound (Ultrasound Bath Grant, Power, 4 times × 10 s) and centrifuged 21,100×g for 15 min at +4 °C. Supernatants were collected and kept at −80 °C until further analysis. EVs were lysed in RIPA Lysis and Extraction Buffer (Pierce, #89900) supplemented with Protease/Phosphatase Inhibitor cocktail (Cell Signalling Technology, 5872S, 1:100) and centrifuged 21,100×g for 15 min at +4 °C. Supernatants were collected and kept at −80 °C until further analysis. Protein concentration was measured using BCA protein assay (Pierce, #23225).
4
Immunoblotting and Coimmunoprecipitation Assays
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For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific), Dithiothreitol, and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). For coimmunoprecipitation assay, cells were lysed in 50 mM Tris, pH 6.8, 0.5% NP-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, and 1× protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with Flag-M2 beads for 2 h at room temperature or with V5 antibodies for 2 h at 4°C, then incubated with Protein A Sepharose 4 Fast Flow and Protein G Sepharose 4 Fast Flow beads (GE Healthcare) for 1 h at 4°C. Immunoprecipitates were subjected to Western blotting. The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 689 STAT2 (Millipore), ISG15 (Santa Cruz Biotechnology), mAb cl.2.1, a gift of E.J. Borden, Cleveland Clinic, Cleveland, OH), V5 (Invitrogen), and Flag (Sigma-Aldrich). Antibodies to phospho-Tyr 701 STAT1, USP18, β-Actin, IFIT1, and AKT were all from Cell Signaling Technology. The chemiluminescence detection reagent was Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Western Lightning Plus-ECL (PerkinElmer).
5
Extraction and Detection of NoV Capsid Protein
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To extract proteins, MNV or mock infected cells were lysed in RIPA buffer (Sigma) supplemented with 100× protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations were measured using the BCA assay (Thermo Fisher). Protein (50 µg) from each sample was separated on an 10% Tris-glycine SDS-polyacrylamide gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Kenilworth, NJ, USA). Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 and incubated with a primary antibody against the NoV capsid protein (Abcam, Cambridge, UK) (ab92976) at a 1:1,000 dilution for 90 min at room temperature. Thereafter, the membrane was incubated for 1 h with an anti-rabbit, HRP-linked secondary antibody (Santa Cruz Biotechnology) at a 1:10,000 dilution. Western blots were developed using a chemiluminescence HRP-substrate (Merck Millipore).
6
Quantification of Hepatic Cytochrome P450s
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PB was purchased from Mallinckrodt (St. Louis, MO). Brij 98 was purchased from Acros Organics (distributed by Thermo Fisher (Fair Lawn, NJ)). The 100 × protease/phosphatase inhibitor cocktail used in the preparation of microsomes was purchased from Cell Signaling Technology (Danvers, MA). The protease inhibitor used in the Brij 98-mediated solubilization of microsomes was the "Complete" EDTA-free tablet from Sigma (St. Louis, MO). The primary antibodies used were as follows: anti-CYP1A1 from Everest Biotech (Oxfordshire, UK) (EB11171) used at 1:500; both anti-CYP3A (PM40) and anti-CYP2E1 (PA26) from Oxford Biomedical (Rochester Hills, MI) used at 1:1000 and 1:4000, respectively; anti-CYP2D6 (MBS821765) from MyBioSource (San Diego, CA) used at 1:1000. Protein standards used for westerns were purified rabbit CYP1A2 (Backes et al., 1998) ; purified, recombinant rabbit CYP2E1 (Cheng et al., 2004) ; purified N-truncated, recombinant CYP3A4 (Moutinho et al., 2012) ; and CYP2D6 human supersomes (Gentest-Corning). All other chemicals of reagent quality that were used were purchased from Sigma.
7
Western Blotting of Immune Signaling Proteins
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Cells were lysed in RIPA buffer (Thermo Fisher Scientific) with 1x Protease/Phosphatase inhibitor cocktail (Cell Signaling Technology). Quantified protein was loaded onto Mini-PROTEAN TGX Precast Gels (Bio-Rad) and resolved using SDS-PAGE and wet transfer onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies (see also dilutions, catalog number and where available clone numbers): STAT1 (1:1,000, #9175, 42H3, Cell Signaling Technology/CST, RRID:AB_2197984), IRF1 (1:1,000, #GTX129134, Source Bioscience, RRID:AB_2885905), Jak1 (1:1,000, #29261, E3A6M, CST, RRID:AB_2798972), GAPDH (1:1,000, #5174, D16H11, CST, RRID:AB_10622025). Blots were developed using chemiluminescence with digital capture of images performed using a Fusion FX6XT digital imaging system (Vilber Lourmat).
8
Western Blot Analysis of Primary CD4+ T Cells
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Primary CD4+ T cells were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 1x protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Lysates were incubated with DTT and NuPAGE LDS sample buffer (Invitrogen). Lysates were run on 12% Criterion TGX gels (BioRad) and transferred to Amersham Hybond polyvinylidene difluoride (PVDF) blotting membranes (Cytiva). Western blots were blocked in 5% BSA in TBS 0.1% Tween and washed in TBS 0.2% Tween. Western blots were incubated with secondary antibody in 5% milk in TBS 0.1% Tween. Western blot chemiluminescence was detected with SuperSignalTM West Femto Substrate (Thermo Scientific). Imaging of the Western blot bands was performed using AlphaView software (ProteinSimple). The following antibodies were used for immunoblots: α-ISG15 (clone F9, 1:500, Santa Cruz Biotech, Cat# sc-166755), α-MX1 (1:1000, Abcam, ab95926), α-GAPDH (clone 6C5, 1:10,000, Sigma Aldrich, MAB374).
9
Western Blot Analysis of Osteogenic Markers
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The cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Delaware, CA, USA) containing a 1x Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The protein content was measured with a protein assay kit (Pierce, Hercules, CA, USA). Protein samples (15 μg), together with a protein marker (Precision Plus Protein Western C Standards; Bio-Rad), were separated on 12% Mini-Protean TGX gels (Bio-Rad, Richmond, CA, USA) for 30 min at 200 V. The separated gels were transferred to a polyvinylidene fluoride (PVDF) membrane for 3 min using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots with Runx2), ALP, OSX, and β-actin (ACTB) were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) as specified by the antibody manufacturer (anti- Runx2) [Abcam, Tokyo, Japan], anti-ALP [Abcam], anti-Sp7/osterix [OSX; Abcam], and anti- ACTB [Bio-Rad] primary antibodies; and horseradish peroxidase-conjugated anti-mouse secondary antibody [Bio-Rad]). The incubated membranes were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent; Cell Signaling Technology). The band density was quantified using the NIH-Image J software and normalized to that of ACTB on day 0.
10
Western Blot Analysis of EGR1 Expression
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Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with 1X Protease/Phosphatase Inhibitor Cocktail (Cell Signaling, Danvers, MA, United States) and separated on 4–20% gradient SDS/PAGE (Novex). Proteins were transferred on nitrocellulose membranes and incubated with primary antibodies overnight at 4°C. The primary antibodies used for Western blotting were rabbit anti-EGR1 (#4153S; Cell Signaling), rabbit anti-GAPDH (#5174S; Cell Signaling) and rabbit anti-β-actin (#4970S; Cell Signaling). The secondary antibody used was HRP linked anti-rabbit IgG (#7074P2; Cell Signaling). Chemiluminescence (ECL) was used to detect protein abundance.
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