Cells were lysed in RIPA buffer (Fisher Scientific, Pittsburgh, PA, USA) in the presence of Xpert protease inhibitor cocktail and Xpert phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA). Proteins were separated by 4–15% Mini-PROTEAN TGX precast gel (Bio-rad, Hercules, CA, USA) and transferred to a nitrocellulose membrane. The antibodies tested included the anti-Erk1/2 antibody; anti-phospho-Erk1/2 (Y202/Y204) antibody; anti-Akt antibody; anti-phospho-Akt (S473) antibody; anti-phospho-Akt (T308) antibody; anti-β-catenin antibody; anti-phospho-β-catenin (S33/S37/T41) antibody; anti-FoxO1 antibody; anti-FoxO3a antibody; anti-phospho-FoxO1 (T24) antibody; anti-phospho-FoxO3a (T32) antibody; anti-β-actin antibody. All the antibodies were purchased from Cell Signaling (Cell Signaling, Danvers, MA, USA).
Xpert protease inhibitor cocktail
Manufactured by GenDEPOT
Sourced in United States
The Xpert protease inhibitor cocktail is a laboratory reagent designed to inhibit the activity of proteases, enzymes that break down proteins. It is intended for use in research applications where the preservation of protein integrity is important.
Automatically generated - may contain errors
Lab products found in correlation
Xpert phosphatase inhibitor cocktail, by GenDEPOT (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ecl chemiluminescence system, by Cytiva (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Chemiluminescence method, by GE Healthcare (1 mentions)
Bioruptor plus sonication device, by Diagenode (1 mentions)
Epitect chip oneday kit, by Qiagen (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g for immunoprecipitation, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Tgx fastcast acrylamide kit, by Bio-Rad (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Vectashield hardset antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Ionomycin calcium salt, by Merck Group (1 mentions)
Jetprime, by Avantor (1 mentions)
17 protocols using xpert protease inhibitor cocktail
1
Western Blot Analysis of Signaling Proteins
2
Whole Mouse Brain Lysate Preparation
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For the preparation of brain lysates, fresh whole mouse brain tissues were lysed using a sonicator (Bioruptor® Plus sonication device; Diagenode, UK) in 200 µl of ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Park et al., 2017 (link)) with Xpert protease inhibitor cocktail (GenDEPOT, USA). Total proteins were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, Sweden). The membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Protein bands were visualized using the chemiluminescence method (GE Healthcare, UK).
3
Chromatin Immunoprecipitation Analysis
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Enrichment of the transcription factor (TF) on the target gene promoters was investigated by ChIP assay using the EpiTect ChIP OneDay Kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s instructions with minor modifications. Briefly, formalin-fixed cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, and 50mM Tris, pH 8.1) including an Xpert protease inhibitor cocktail (GenDEPOT, Katy, TX, USA). To shear chromatin to an average length of about 200–500 bp, cell lysates were sonicated using a sonicator (Branson-Emerson, MO, USA). Sheared chromatin was pre-cleared with protein agarose beads at 4 °C for 1 h and then incubated with a primary antibody (2 μg) overnight at 4 °C. The protein–antibody complex was pulled down with the protein agarose bead at 4 °C for 1 h and sequentially washed with a low-salt solution, a high-salt solution, LiCl solution, and Tris-EDTA solution twice. DNA was eluted after incubating with elution buffer (1% SDS and 0.1 mol/L NaHCO3) containing protease K at 45 °C for 30 min using DNA-binding beads and columns. TF binding on the target gene promoters was analyzed by qPCR. Primer sets for ChIP assay are shown in Table S2 .
4
Immunoprecipitation and Western Blotting
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Antibodies used in this study are reported in Supplementary Table S3 . Reagents and chemicals include: Ionomycin calcium salt (Sigma # I3909), Protein G-Agarose (Sigma #11243233001), Streptavidin Sepharose High Performance bead (Sigma #17-5113-01), Calmodulin Sepharose 4B (Sigma #17-0529-01), Dynabeads™ Protein G for Immunoprecipitation (Thermo Fisher Scientific #10004D), jetPRIME®, DNA and siRNA transfection reagent, Polyplus-transfection® reagent (VWR #89129-922), TRIzol™ Reagent (Thermo Fisher Scientific #15596018), Quantitect reverse transcriptase (Qiagen #205313), Xpert Protease inhibitor cocktail (GenDEPOT #P3100-100), Xpert Phosphatase inhibitor cocktail (GenDEPOT #P3200-020), Penicillin-Streptomycin (GenDEPOT #CA005-100), 10× PBS Buffer (GenDEPOT #P2100-100), DMEM, High Glucose with L-Glutamine (GenDEPOT #CM002-050), Opti-MEM™ Reduced Serum Medium (Thermo Fisher Scientific #31985070), FBS Opti-Gold, Us Origin (GenDEPOT #F0900-050), iTaq Universal SYBR® Green Supermix (Bio-Rad #1725124), Immun-Blot PVDF Membrane (Bio-Rad #1620177), TGX™ FastCast™ Acrylamide Kit (Bio-Rad #161-0173, #161-0175), Precision Plus Protein™ Dual Color Standards (Bio-Rad #1610394), Blotto, non-fat dry milk (Santa-Cruz # sc-2325), SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific #34076), VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vectorlabs #H-1500).
5
Western Blot Protein Analysis Protocol
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Cells were lysed in RIPA buffer containing Xpert Protease Inhibitor cocktail (GenDEPOT, Katy, TX, USA). Lysates were centrifuged 12,000 rpm at 4˚C for 30 min and then the supernatants were collected and used. Proteins were separated by 8% SDS-polyacrylamide electrophoresis and transferred to PVDF membrane (Bio-rad, Hercules, CA, USA). The membranes were blocked by 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) buffer for 1 h at room temperature and probed with primary antibodies (1:1,000) overnight at 4˚C. Membranes were washed in TBS-T buffer and incubated for 1 h at room temperature with HRP-conjugated specific secondary antibodies. After membranes were washed in TBS-T buffer, blots were developed using the ECL system (Amersham, Buckinghamshire, UK) and detected by ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA).
6
Western Blot Analysis of Protein Extracts
7
Protein Extraction and Immunoblotting Protocol
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Cells were washed once with phosphate-buffered saline (PBS) and lysed by ice-cold radioimmunoprecipitation assay (RIPA) buffer containing Xpert Protease Inhibitor Cocktail (GenDEPOT, Inc., Barker, TX, USA) and 0.5 mM sodium orthovanadate (Na3VO4). Lysates were centrifuged (12,000 g) at 4 °C for 30 min and then supernatants were used for immunoblot analysis. Proteins were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked by 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and probed with primary antibodies followed by HRP-conjugated specific secondary antibodies. Immunoreactive blots were developed using ECL chemiluminescence system (Amersham, Buckinghamshire, UK) and exposed to blue X-ray film (AGFA, Mortsel, Belgium).
8
Western Blot Protein Analysis
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Cells were lysed in radioimmunoprecipitation buffer containing Xpert protease inhibitor cocktail (GenDEPOT, Barker, TX, USA), sodium fluoride (1mM), β-glycerophosphate (1mM), sodium orthovanadate (1mM), and sodium pyrophosphate (2.5mM). After incubation for 30 min on ice, cell lysates were collected by centrifugation at 15,000g for 30 min. Protein concentrations were determined using a bicinchoninic acid assay kit (Thermo, Rockford, IL, USA). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Immunoreactive proteins of interest were visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences). Equal protein sample loadings in gels were verified by β-actin immunoblotting. Immunoblot intensities were quantified by densitometric analysis (Image J, rsb.info.nih.gov/ij).
9
Galectin-3 Regulation of Nuclear and Cytoplasmic Extracts
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Nuclear and cytoplasmic extracts were prepared from SKOV3 and A2780 cells after treatment with galectin-3 siRNA and transfection with galectin-3 overexpression vectors. Cells were lysed in Buffer A (10 mM HEPES (pH 7.9), 1.5 mM Mgcl2, 10 mM KCl, 1 mM DTT, 0.2 mM PMSF, 0.1% NP-40) containing a Xpert protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) and phosphatase inhibitor (NaF, Na3VO4), incubation on ice for 15 min. The cell lysate was centrifuged for 10 min at 850 G at 4°C and discard the supernatant. Next, resuspend the pellet with Buffer C (20 mM HEPES (pH 7.9), 25 % Glycerol, 0.42 M Nacl, 0.2 mM EDTA, 1.5 mM Mgcl2, 1 mM DTT, 0.2 mM PMSF) and vortex for 15 sec. Incubate the cell lysate for 30 min on ice and vortex every 10 min for 15 sec. After incubation the cell lysate were centrifuged for 10 min at 13,200 rpm at 4°C and collect the supernatant (Nuclear). Western blot analysis was performed as described previously [39 (link)] with nuclear fraction protein samples.
10
Western Blot Analysis of PPARγ, Adiponectin, and HSP90
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Cells were lysed with the use of the Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) containing an Xpert protease inhibitor cocktail (GenDEPOT, USA), and were then centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were separated by precast gels (Bio-Rad Laboratories) using a running buffer, and were subsequently electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h in Tris-buffered saline with Tween 20 (TBST; containing 20 mM Tris-HCl, 150 mM NaCl, 0.2% Tween 20, pH 7.4) supplemented with 5% bovine serum albumin, and were then incubated with an anti-PPARγ antibody (Cat. No. 2443; Cell Signaling Technology, USA), an anti-adiponectin antibody (Cat. No. MA1-054; Thermo Fisher Scientific), and an anti-HSP90 antibody (Cat. No. SC-13119; Santa Cruz Biotechnology, USA), at 4°C overnight. After washing with fresh TBST, the membrane was incubated with secondary antibodies conjugated with horseradish peroxidase specific to rabbit or mouse IgG (1:5,000 dilution; Bio-Rad Laboratories), and were visualized by using the ECL system (Bio-Rad Laboratories) followed by an iBright CL1500 imaging system (Thermo Fisher Scientific).
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