Cell lysates were prepared in RIPA lysis buffer (50 mM Tris, pH8, 150 mM NaCl, 1% Triton-X, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and protease inhibitor cocktail (Roche)), separated by SDS-PAGE and transferred to nitrocellulose membranes using the Transblot Turbo Transfer System (Bio-Rad). Membranes were labelled with the indicated primary and secondary antibodies and, imaged and quantified on the LI-COR Odyssey. Where applicable, quantification of protein bands of interest were normalized relative to GAPDH levels.
Protease inhibitor cocktail
Manufactured by Bio-Rad
Sourced in United States
Protease inhibitor cocktail is a pre-formulated solution designed to inhibit the activity of various proteases. It is commonly used in research applications to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Iscript reverse transcription supermix for qrt pcr, by Bio-Rad (4 mentions)
Phosphatase inhibitor cocktail, by Merck Group (4 mentions)
Alamar blue, by Thermo Fisher Scientific (4 mentions)
Pageruler plus prestained protein, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Accutase solution, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
β actin, by Merck Group (3 mentions)
Pgfp c shlenti, by OriGene (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Taqman advanced mirna cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
28 protocols using protease inhibitor cocktail
1
Protein Quantification by Western Blotting
2
Immunoprecipitation of Glomerular Proteins
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Immunoprecipitation was performed basically according to the method previously reported (Hashimoto et al. 2007 ; Otaki et al. 2008 ). In brief, glomerular lysate solubilized with 1% Triton X‐100 with a protease inhibitor cocktail (Bio‐Rad) was incubated with an anti‐CN‐A antibody or normal rabbit serum at 4°C overnight and precipitated with Dynabeads Protein G (Invitrogen, Carlsbad, CA). The tube was placed in the magnet, and the supernatant was removed. The Dynabeads–antibody–antigen complex was washed five times with PBS containing 0.1% Triton X‐100, and then the antigen was eluted with the SDS‐PAGE sample buffer. The elution fractions were separated by SDS‐PAGE followed by immunoblotting with a rabbit anti‐nephrin antibody or a rabbit anti‐ZO‐1 antibody. The antigen of another tubes was eluated with the SDS‐PAGE sample buffer without mercaptoethanol. The elution fractions were separated by SDS‐PAGE followed by immunoblotting with a rabbit anti‐podocin antibody. Alkaline phosphatase‐conjugated antibody was used as a second antibody.
3
Preparation of Cell Extracts for Immunoblotting
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For preparation of cell extracts, 50 ml 6 days old cultures were concentrated by centrifugation (5000 × g, 10 min), resuspended with 0.5 ml TE (10 mM Tris-HCl pH 8., 1 mM EDTA) and freshly prepared protease inhibitor cocktail (Sigma, P8465-5ML) was added to 0.86 mg/ml. Glass beads (0.2 g) were added for breakage by mixer mill (Retch MM400) at a frequency of 30 s−1 for 2 min in pre-chilled holders (5 times, 1 min incubation on ice between the cycles). Cell lysates were centrifuged (835 × g, 5 min, 4 °C) to pellet the glass beads. Cell lysates were transferred to a fresh Eppendorf tube, diluted with TE supplemented with protease inhibitor cocktail and 2.3 µl from diluted extracts were spotted onto TransBlot Turbo nitrocellulose membrane (Bio-Rad) and air dried for 5 min. All following procedures were performed at room temperature: Blocking was done for 1 h in 0.1% bovine serum albumin in TBST (20 mM Tris-HCl (pH 8.0) and 0.05% Tween20). Incubation with anti-FLAG (ab1162, Abcam; 1:2000 diluted in blocking solution) was performed for 1 h following three washes in TBST for 5 min each. Incubation with secondary antibodies (goat anti rabbit IgG, 170-6515, Bio-Rad; 1:5000 diluted in blocking solution) was done for 1 h following washes as above with extension of last wash to 15 min and signal detection using SuperSignal West Pico kit (Thermo Scientific, 34080).
4
Exosome Protein Extraction and Western Blot
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For total protein extraction from exosomes, lysis was performed with RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail and Laemmli buffer (Bio-Rad). The total protein concentration was quantified using a BCA protein assay kit (Thermo Scientific) following the manufacturer’s instructions. Briefly, approximately, 30 μg of protein extracts were electrophoresed on a 4-15% Mini protean pre-cast polyacrylamide gel (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk (Cell Signaling) for 1 hour and immunoblotted overnight at 4 °C with primary antibodies to CD63 (ExoAB-CD63A-1 SBI system Biosciences), CD81 (ab59477, Abcam), Hsp70 (EXOAB-Hsp70A-1), CD91 (ab20384, Abcam) and GAPDH (ab8245, Abcam). After incubation, anti-rabbit (7074S, Cell Signaling Technology/EXOAB-HRP) or anti-mouse (ab97040, Abcam) secondary antibodies were applied for 1 hour. The membrane was then visualized by Clarity western ECL substrate (Bio-Rad). Images were acquired using the Bio-Rad Chemidoc MP Imaging system.
5
Protein Extraction and Purification
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TAA, formaldehyde, urea, CHAPS, DTT, iodoacetamide, methanol, ethanol, and sodium thiosulfate were purchased from Sigma-Aldrich (Darmstadt, Germany). Bis-acrylamide solution and protease inhibitor cocktail were purchased from Bio-Rad (Hercules, CA, USA). High-purity glacial acetic was purchased from J.T Baker (Loughborough, England). All other chemicals used in this study were the highest grade commercially available.
6
Protein Extraction from Pig Tissues
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The frozen LM tissues (100 mg) from Shaziling pigs and Yorkshire pigs were crushed in a mortar containing liquid nitrogen and were then sonicated for 10 s using a Sonoplus (Bandelin Electronic, Berlin Germany). The crushed tissue was homogenized in 1 mL of cold dissolution buffer containing 7 M urea, 2 M thiourea, 1 % dithiothreitol (DTT), 4 % (w/v) CHAPS, 20 μL protease inhibitor cocktail (BBI, Canada), and 2 % (v/v) pharmalyte (pH 3–10; BioRad, Hercules, CA, USA). The homogenate was centrifuged at 15,000 × g, for 20 min at 4 °C. The supernatant fraction was filtered and kept at −80 °C for subsequent analysis. The total protein content was determined using a Bradford assay kit (Bio-Rad).
7
Baicalein Modulation of STAT Signaling
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Naive CD4+ T cells (1 × 107 cells/well) were cultured with 5 μmol/L baicalein in 12-well plates in the presence 10 μg/mL plate-bound anti-CD3 mAb and 2 μg/mL soluble anti-CD28 mAb for 48 h. The cells were lysed with RIPA buffer including protease inhibitor cocktail (Bio-Rad, Richmond, CA, USA). Quantified proteins (20 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were incubated with specific primary [total- and phospho-STAT1 (1:1000), STAT3 (1:2000), STAT5 (1:1000), and STAT6 (1:1000)] (Cell Signaling Technology, Beverly, MA, USA) and secondary antibodies (1:3000) (Bio-Rad). The membranes were subjected to an enhanced chemiluminescence reaction and signals were detected using an AE9300 instrument (ATTO, Tokyo, Japan).
8
Activation and Inhibition of MMP9-PARP1 Interaction
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With 1 mM APMA, 10 ng of human pro-MMP9 (Enzo Life Sciences, Farmingdale, NY) was activated at 37°C for 2 h. To the activated MMP9, 100 ng of human PARP-1 [poly-(ADP-ribose)-polymerase 1; Sino Biological, Beijing, China] was added with or without increasing concentrations of 40-mer oligonucleotides in assay buffer (25 mM Tris–HCl, 100 mM NaCl, 2.5 mM CaCl2, 750 nM ZnCl2, and 0.025% Brij-35, pH 7.0) for 2 ½ h at 37°C. Reactions were stopped in RIPA buffer containing a protease inhibitor cocktail (Bio-Rad).
9
Inhibition of CHEK1 in Cell Lines
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The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10565-018), fetal bovine serum (Gibco, 10082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), phosphatase inhibitor cocktail (Sigma, P0044), protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCHEK1 lentivirus particles (pGFP-C-shLenti, Origene, TR320302), CHIR-124 (Selleckchem, S2683), anti-CHEK1 (Novus Biologicals, NB100-464, rabbit, used for WB and IHC), anti-RAD54L (Novus Biologicals, NBP2-33916, rabbit, used for WB), and β-Actin (Sigma, A5316, mouse, used for WB).
10
Western Blot Analysis of RBPMS Protein
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Cells were collected and washed a few times with phosphate buffer saline (PBS) and stored at −80 °C until use. Cell pellets were lysed with ice-cold lysis buffer (1% Triton X, 150 mmol/L NaCl, 25 mmol/L Tris HCl, 0.4 mmol/L NaVO4, 0.4 mmol/L NaF, and a protease inhibitor cocktail from Sigma), and the total protein concentration was determined using Bio-Rad DC Protein Assay reagents (Bio-Rad, Hercules, CA, USA). The protein samples were separated via SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked in either 5% non-fat dry milk (BioRad, Hercules, CA, USA) or 5% BSA (HyClone) Sigma-Aldrich, St. Louis, MO, USA and probed with primary antibodies (anti-RBPMS, 1:1000 dilution) or anti-β-actin (Sigma, St. Louis, MO, USA; 1:10,000 dilution). The membranes were then incubated with mouse or rabbit IgG horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling, 1:5000 dilution) followed by enhanced chemiluminescence and autoradiography. Bands were imaged with a Bio-Rad Gel Doc XR+, and the signal intensity of each band was quantified using Image Lab software (BioRad, Hercules, CA, USA).
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