Recombinant human kallistatin was expressed in Pichia pastoris strain GS115 and purified with a series of chromatographic steps, mainly Phenyl Superose and Heparin Sepharose FF chromatography. The antibodies for nucleolin, kallistatin, CK2, GAPDH, MMP2, Integrin β3, LaminB2 were raised in rabbits and were obtained from Cell Signaling Technologies (Boston, MA). Mouse anti-nucleolin antibodies were obtained from Life Technology (Gaithersburg, MD). PVDF membranes and Immobilon ECL were purchased from Millipore Co (Billerica, MA). Protease inhibitor cocktail was obtained from Amresco Inc. (Solon, OH, USA). Dulbecco's modified Eagle's medium (DMEM), DMEM/F12/ Glutamax and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Endothelial Growth Medium-2 (EGM-2) and Endothelial Basal Medium-2 were purchased from Lonza Inc (Walkersville, ML). Protein A beads were purchased from Sigma-Aldrich (Steinheim, Germany).
Lamin b2
Manufactured by Cell Signaling Technology
Lamin B2 is a structural protein found in the nuclear lamina of eukaryotic cells. It is a component of the nuclear envelope and is involved in the organization and maintenance of the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (8 mentions)
Western ecl substrate mixture, by Bio-Rad (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Ibright fl1000, by Thermo Fisher Scientific (4 mentions)
Cleaved caspase 1, by Cell Signaling Technology (2 mentions)
Ibright image analysis software, by Thermo Fisher Scientific (2 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ripk3, by Cell Signaling Technology (2 mentions)
P mlkl, by Cell Signaling Technology (2 mentions)
P tak1, by Cell Signaling Technology (2 mentions)
C caspase 3, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Integrin β3, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Protein a beads, by Merck Group (1 mentions)
Immobilon ecl, by Merck Group (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
8 protocols using lamin b2
1
Recombinant Kallistatin Purification in Pichia
2
Protein Extraction and Immunodetection
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Cells were plated in 160 cm2 Petri dishes (1.5 × 106 cells/dish) in the culture medium. Cells were lysed using a detergent cocktail (M-PER mammalian extraction buffer) supplemented with protease inhibitors (Halt protease inhibitor cocktail) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail; all from Pierce, Rockford). Protein concentration was measured by the BCA Protein Assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific™) using bovine serum albumin as the standard. Immunodetections used antibodies raised against, phospho-Met (1/1,000), lamin B2 (1/1,000; all from Cell Signaling Technology), PARP-1 (1/150; from Thermofisher Scientific, Life Technologies Europe BV), PAR (1/1,000; R&D systems, Bio-Techne), and β-actin (1/5,000; from Millipore, Temecula). Peroxidase-labeled anti-rabbit IgG antibody (1/5,000) or peroxidase-Duolink® PLA Flow Cytometry antibody (1/5,000; both from GE Healthcare Europe G mbH) were used as secondary antibodies. Bound peroxidase activity was revealed using the SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Relative protein expressions (fold-change) were calculated and normalized relative to β-actin the whole uncropped images of the original western blots from which figures have been derived are submitted in Supplementary materials (Figures S3–S6 ).
3
Regulation of TRAIL and NF-κB in BM-MDSCs
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For the SB conditioned medium studies, BM-MDSCs were plated in 10-cm dishes, and on the following day were incubated with an equal volume of SB cell conditioned medium for 0, 0.5, 1, 2, or 4 hours. BM-MDSC cytoplasmic and nuclear protein lysates were collected using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (#78833; Thermo Fisher) per the manufacturer’s protocol. Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The following primary antibodies were used for immunoblotting analysis: polyclonal TRAIL (ab231265 or ab42121-100; Abcam), IκBα (#4814s; Cell Signaling Technology), phospho-IκBα (Ser32) (#2859s; Cell Signaling Technology), phospho-IκBα (Ser36) (#ab133462; Abcam), NF-κB p65 (#SC-8008; Santa Cruz), Lamin B2 (#12255; Cell Signaling Technology), histone H3 (#4499s; Cell Signaling Technology), and cFLIP (#5634s; Cell Signaling Technology). Membranes were blotted with primary antibody overnight at 4°C at a dilution of 1:1000. Goat anti-mouse or rabbit IgG (H + L)–horseradish peroxidase (HRP) conjugate secondary antibodies were used at a dilution of 1:2000. Proteins were visualized with Clarity and Clarity Max ECL Western Blotting Substrates (Bio-Rad).
4
Western Blot Analysis of Liver Proteins
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Protein was extracted from liver tissue or cell cultures as described [7 (link)]. Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). The buffer contains 1% proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). The Foxo1, p-JNK, JNK, p-Akt, p-β-catenin, β-catenin, Snail, HMGB1, RIPK3, p-MLKL, NLRP3, cleaved caspase-1, Lamin B2, β-actin (Cell Signaling Technology), Gli1 (Santa Cruz Biotechnology), Sonic Hedgehog (Shh), SMO, NEK7, and TCF4 (Abcam) mAbs were used. The membranes were incubated with Abs, and then added Western ECL substrate mixture (Bio-Rad) for imaging with the iBright FL1000 (ThermoFisher Scientific). Relative quantities of protein were determined by comparing to the β-actin expression, using iBright image analysis software (ThermoFisher Scientific).
5
Western Blot Analysis of Protein Extraction
6
Protein Extraction and Western Blot Analysis
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Protein was extracted from liver tissue or cell cultures as described [19 (link)]. The NICD, Bax, Bcl-2, c-caspase-3, TRAF6, p-TAK1 (Ser412), TAK1, p-P65 (Ser536), P65, RIPK3, p-MLKL (Ser345), Lamin B2, β-actin (Cell Signaling Technology) were used. The Western ECL substrate mixture (Bio-Rad) was used to image with the iBright FL1000 (ThermoFisher Scientific). See Additional file 1 : Materials.
7
Western Blot Analysis of Protein Expression
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Tissues or cells were equilibrated in immunoprecipitation assay buffer at 4 °C for 30 min. The supernatant was collected and centrifuged at 12,000 × g for 20 min. After separating proteins by polyacrylamide gel electrophoresis, they were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with antibodies against Caspase 6 (ab185645, Abcam, 1:1000 dilution), NEK7 (sc-393539, Santa Cruz Biotechnology, 1:1000 dilution), NLRP3 (ab270449, Abcam, 1:1000 dilution), cleaved Caspase 1 (C-caspase 1) (#89332, Cell signaling Technology, 1:1000 dilution), NR4A1 (ab153914, Abcam, 1:1000 dilution), SOX9 (ab185966, Abcam, 1:1000 dilution), S100A9 (ab242945, Abcam, 1:1000 dilution), and β-actin (#4970, Cell signaling Technology, 1:2000 dilution). β-actin was used as an internal reference. The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). Lamin B2 (#12255, Cell signaling Technology, 1:1000 dilution) was used as an internal reference of nuclear protein. IBright FL1000 (Invitrogen, Carlsbad, CA, USA) was used to analyze the expression of target proteins. Full and uncropped western blots have been shown in Supplementary Material (2) .
8
Comprehensive Protein Analysis of Liver Tissue
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Protein was extracted from liver tissue or cell cultures as described (19) . The NICD, Bax, Bcl-2, c-caspase-3, TRAF6, p-TAK1, TAK1, p-P65, P65, RIPK3, p-MLKL, Lamin B2, β-actin (Cell Signaling Technology) were used. The Western ECL substrate mixture (Bio-Rad) was used to image with the iBright FL1000 (ThermoFisher Scienti c). See Supplementary Materials.
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