HSCs were treated with or without 100U IFN-γ for 48 hr. For negative controls, CD8+ T cells were isolated from WT C57BL/6 mouse splenocytes using a mouse CD8+ T cell isolation kit (Miltenyi Biotec, San Diego, CA), and for positive controls, mouse splenocytes after B cell depletion using a mouse B cell isolation kit (StemCell, Vancouver, Canada) were used. Cell lysates were prepared using NP-40 buffer (Sigma, MI) and total protein concentrations were determined by using a BCA protein concentration assay kit (Pierce, IL) following manufacturer provided protocols. Blotted membrane was incubated with 1 µg/ml of the sheep anti-GARP Ab (R&D, Minneapolis, MN) at 4°C overnight, then incubated with 2 µg /ml of the HRP-conjugated Rabbit anti-sheep IgG (Southern Biotech, Birmingham, AL) for 30min at room temperature. After washing, ECL western blotting regent (GE Healthcare Bio-Sciences, Pittsburg, PA) was applied for the detection. The same membrane was stripped and probed again using an anti-mouse actin antibody to serve as a loading control.
Np 40 buffer
Manufactured by Merck Group
Sourced in United States
NP-40 buffer is a non-ionic detergent used in various laboratory applications. It is commonly used as a cell lysis agent and for the solubilization of membrane proteins. The buffer helps to disrupt cell membranes and extract proteins while maintaining their native structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor, by Merck Group (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Vegfr2 55b11, by Cell Signaling Technology (2 mentions)
Nonfat milk powder, by Merck Group (2 mentions)
Mouse cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Mouse b cell isolation kit, by STEMCELL (1 mentions)
Gapdh, by Merck Group (1 mentions)
Col 1, by Abcam (1 mentions)
Col 3, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Prime western blotting detection reagent, by GE Healthcare (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
9 protocols using np 40 buffer
1
GARP Expression in Mouse HSC
2
Evaluating Collagen Expression in HFB Cells
3
Endogenous Protein Immunoprecipitation
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For endogenous MYCN and SNRPD3 immunoprecipitation assays, SK-N-BE(2)-C and KELLY cells were lysed in cold BC100 (Sigma-Aldrich) or NP40 buffer (Sigma-Aldrich), respectively, supplemented with protease inhibitor (Sigma-Aldrich). Five percent of the cell extract was saved as the input, the remainder (750 μg of total protein) was incubated with either 10 μg MYCN-specific antibody (Merck Millipore Millipore, VIC, Australia) or 5 μg SNRPD3-specific antibody (Abcam, ab157118) or control IgG antibody (10 μg for mouse (Santa Cruz Biotechnology) or 5 μg for rabbit (Cell signalling)) and A/G PLUS agarose beads (Bio-Strategy) at 4 °C overnight. After 3 washes with the lysis buffer, the bound proteins were eluted by boiling with SDS sample buffer. Bound proteins were resolved by SDS-PAGE.
4
Western Blot Analysis of VEGFR2 and ASM
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Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4 °C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Cell Signaling Technology) antibody overnight at 4 °C, rinsed and incubated in peroxidase-conjugated secondary antibodies (Santa Cruz, Heidelberg, Germany) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science). VEGFR2 and ASM expression were normalized to β-actin abundance.
5
Immunoprecipitation and Deacetylation Assay
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Cells were lysed in NP-40 buffer containing 50 mM Tris-HCl (pH 7.5) (Sigma, St Louis, MO, USA), 150 mM NaCl (Sangon, Shanghai, China), 0.5% Nonidet P-40 (Sigma), 1 μg/ml aprotinin (Sigma), 1 μg/ml leupeptin (Sigma), 1 μg/ml pepstatin (Sigma), 1 mM Na3VO4 (Sigma) and 1 mM PMSF (Sigma). For immunoprecipitation, 500 μl of cell lysate was incubated with HA antibody (provided by the Zhao lab of Fudan University) for 3 h at 4 °C with rotation. Then, 30 μl of Protein A Agarose (Millipore, Billerica, MA, USA) was added for 12 h at 4 °C with rotation, and the beads were washed three times with lysis buffer before proteins were dissolved in loading buffer. Deacetylation assays were carried out in the presence of 5 μg enzyme and 0.3 μg peptide in 30 μl reaction buffer [30 mM HEPES (Sigma), 0.6 mM MgCl2 (Sangon), 1 mM DTT (Sigma), 1 mM NAD+ (Sigma), 10 mM PMSF (Sigma)]. The deacetylation reaction was incubated for 2 h at 37 °C before the mixture was desalted by passing it through a C18 ZipTip (Millipore). The desalted samples were analyzed using a MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Grand Island, NY, USA). The acetylated peptide used in the assay was TRKDYPAAK (Ac) RVKLDSVR (Glssale, Shanghai, China).
6
KRIT1 Phosphorylation in HeLa and HPAECs
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GFP–KRIT1-transfected HeLa cells treated with PMA or BIM+PMA were lysed in NP-40 buffer (Sigma) containing protease and phosphatase inhibitors (P8340 and P2850, respectively; Sigma). GFP–KRIT1 was immunoprecipitated from cell lysates using the rabbit polyclonal anti-GFP antibody (ab290, Abcam), and analyzed by western blotting with pan-phospho-Ser/Thr antibody ab17464 (1:1000). Western blotting analysis was performed as previously described (Balzac et al., 2005 (link)).
mCherry–KRIT1-expressing HPAECs treated with PMA, BIM and BIM+PMA were lysed in buffer containing 20 mM HEPES-KOH pH 7.5, 1.5 mM MgCl2, 5 mM KCl, and protease and phosphatase inhibitors, supplemented with 1% Triton X-100. KRIT1 was immunoprecipitated (Mab15.B2, Millipore, Burlington, MA) from total lysate and blotted with pan-phospho-Ser/Thr antibody 22A (1:1000).
mCherry–KRIT1-expressing HPAECs treated with PMA, BIM and BIM+PMA were lysed in buffer containing 20 mM HEPES-KOH pH 7.5, 1.5 mM MgCl2, 5 mM KCl, and protease and phosphatase inhibitors, supplemented with 1% Triton X-100. KRIT1 was immunoprecipitated (Mab15.B2, Millipore, Burlington, MA) from total lysate and blotted with pan-phospho-Ser/Thr antibody 22A (1:1000).
7
Confirming MDA5-CIV Protein Interaction
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To confirm the interaction between MDA5 and CIV proteins, 293T cells were transfected with the 3xFlag-tagged NS1, PA, PB1, PB2, NP, and MYC-tagged MDA5 plasmids, harvested 48 h post-infection (pi), washed three times with cold PBS, and lysed with NP-40 buffer (Sigma–Aldrich, 127087–87-0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime, ST506) for 1 h at 4 °C. Clarified extracts were precleared with protein A/G beads (Santa Cruz Biotechnology, sc-2003) plus an anti-Flag monoclonal antibody for 4 h, washed with NP-40 buffer, boiled in sample buffer, and subjected to sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE). This was followed by immunoblot analysis with anti-Flag and anti-MYC antibodies.
8
Shedding Light on Angiogenic Factors
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Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4°C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Acid sphingomyelinase deactivation post-ischemia/ reperfusion promotes angiogenesis/ 26 Amnis ImagestreamX flow cytometry of sEVs sEVs were quantified with an ImageStreamX MkII instrument (Merck Millipore) as described previously 55 after CD9-FITC (MEM-61; Exbio, Vestec, Czech Republic) and CD63-APC (MEM-259; Exbio) antibody staining. All samples were appropriately diluted in order to avoid coincidence or swarm detection. Data analysis was performed using Amnis IDEAS software (version 6.1).
9
IgG-Stimulated Signaling Pathway Analysis
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Human endothelial cells were serum-starved overnight before IgG stimulation. Cells were lysed in 1% NP40 buffer (Sigma-Aldrich, St. Louis, MO) containing Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and migrated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis gels. Proteins were transferred to Hybond-P PVDF membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA) and incubated with Abs against pERK1/2 (T202/Y204; D13.14.4), pAkt (S473; 193H12), pMEK1/2 (S217/S221; 41G9), pS6K (T389; 108D2), pS6RP (S235/S236; D57.2.2E), horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology, Danvers, MA), actin (I-19), and horseradish peroxidase-conjugated anti-goat (Santa Cruz Biotechnology, Dallas, TX). Chemiluminescence from ECL prime reagents (GE Life Sciences) was evaluated by the Fusion Solo S (Vilber Lourmat, Marne-La-Vallée, France) and analysis was performed with Image J and Multigauge (Fujifilm Minato, Tokyo, Japan). The optical density of phosphorylated protein was normalized to actin levels. Phosphorylation of signaling proteins was assessed using the Pathscan Intracellular Signaling Array Kit (Ref 7323S; Cell Signaling Technology).
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