Cells were lysed with lysis buffer supplemented with protease inhibitor cocktail (Roche). Total protein in cell lysates were incubated with 2 μg anti‐STAT3 or anti‐SHP‐1 antibody (Supplementary Table S3 ) at 4°C for 2 hours, and with 20 μL A/G PLUS‐Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After incubation, the beads were separated from the lysis buffer, washed 5 times in cold NP‐40 buffer (Beyotime), and then subjected to western blotting.
Np 40 buffer
Manufactured by Beyotime
Sourced in China, United States
NP-40 buffer is a non-ionic detergent commonly used in cell lysis and protein extraction procedures. It is effective in solubilizing membrane proteins while preserving protein-protein interactions. The buffer's composition and concentration can be adjusted to suit specific experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Ab6721, by Abcam (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Anti flag m2 affinity gel, by Merck Group (3 mentions)
Ab236632, by Abcam (3 mentions)
Polyvinylidene fluoride (pvdf), by Thermo Fisher Scientific (3 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Abcam (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Eyoecl plus, by Beyotime (2 mentions)
Quantity one software version 4, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Mg132, by MedChemExpress (2 mentions)
Ab205718, by Abcam (2 mentions)
82 protocols using np 40 buffer
1
Immunoprecipitation and Western Blotting
2
Bim Protein Immunoprecipitation and Ubiquitin Analysis
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Cells were lysed in NP-40 buffer (Beyotime) supplemented with phosphatase and protease inhibitors, and the lysates were immunoprecipitated at 4 °C overnight with the SureBeads protein A (Bio-Rad) conjugated with the Bim antibody (Cell Signaling). Precipitates were washed three times with lysis buffer. Protein levels were evaluated using immunoblotting analysis with ubiquitin antibody (Thermo).
3
Western Blot Analysis of Protein Lysates
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Cells were lysed in NP40 buffer (Beyotime) containing 1% 100 mM PMSF (Beyotime) for 30 min, and then boiled under 1Xloading buffer at 105 ℃ for 10 min. An equal amount of protein was loaded on 10% SDS-PAGE gels and electrically transferred to a nitrocellulose filter membrane after separation. The membrane was blocked with 8% de-skimmed milk (4 g milk powder with 50 ml TBS Tween-20 buffer) for 1–2 h and then incubated with primary antibodies at 4 °C overnight. Then, after using TBS-Tween washing membrane three times for 10 min each time, the band was incubated with anti-rabbit IgG or anti-mouse IgG at room temperature for 60 min. All antibodies used are shown in Additional file 2 : Table S2. The images were created by adding enhanced chemiluminescence (NCM Biotech) with image laboratory software.
4
Identification of SIRT5-Interacting Proteins
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Cells were lysed with 1% NP-40 buffer (P0013F, Beyotime) with freshly added proteinase and phosphatase inhibitor cocktail. Then the lysate was incubated with Flag Fab-Trap™ Agarose (ffa, Chromotek, Proteintech) or Anti-GFP Beads (L-1016, Biolinkedin, Shanghai, China) with gentle rotation overnight at 4 °C. The captured complexes were washed, boiled at 95 °C in SDS sample buffer, and detected by SDS-PAGE. To investigate the SIRT5-interacting proteins, the gels were stained with coomassie brilliant blue (CBB) and cut for mass spectrometry analysis, which was performed by PTM Bio. The interaction between HINT1 and SIRT5, as well as the acetylation or succinylation of HINT1, were further validated by Western blot using corresponding antibodies.
5
Eltrombopag Regulation of VEGF-A and MMP9
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Cells were seeded in 6-well plates and treated with eltrombopag (10 μmol/L) for 24 h. Cells were collected and lysed with NP40 buffer (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Protein concentrations were determined with the Enhanced BCA Protein Kit (Beyotime Biotechnology). Western blots were performed as described34 using antibodies (Santa Cruz Biotechnology) against VEGF-A (sc-152), MMP9 (sc-6840), HuR (sc-5261), and β-actin (sc-8432). Detections were analyzed with Azure Biosystem (Azure c600, Azure Biosystem™, Dublin, CA, USA).
6
Protein Interaction Analysis by Western Blotting and Co-Immunoprecipitation
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For western blotting analysis, cells were lysed in RIPA buffer (P0013B, Beyotime) containing protease and phosphatase Inhibitor cocktail, EDTA free (78441, Thermo Fisher Scientific) for 30 min on ice followed by pelleting of insoluble material by centrifugation. Protein concentration was determined using a BCA assay. For co-immunoprecipitation experiments, 10 million cells were lysed in 1 ml NP40 buffer (P0013F, Beyotime) containing protease and phosphatase Inhibitor cocktail, EDTA free, for 30 min on ice. After concentration, supernatants were incubated with Rabbit anti-DENR, Rabbit anti-JAK2, or Rabbit normal IgG for 4 h at 4 °C, followed by addition of 25 µl protein A/G beads (B23201, Bimake) and incubation for a further 2 h. After five washes in NP40 buffer, beads were eluted in 100 μl IgG elution buffer, PH2.0 (21028, Thermo Fisher Scientific) and neutralized with 15 μl 50 mM NaOH.
7
Protein and Cytokine Analysis of Primary Cells
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Primary cell cultures were homogenized in NP40 buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Roche). The cell lysates were subjected to Western blotting using the anti‐IRF4 (1:500, sc‐6059, Santa Cruz), anti‐TNF‐α (1:500, AF‐510‐NA, R&D), anti‐MBP (1:500, MAB382, Chemicon), anti‐EZH2 (1:500, ab3748, Abcam), and HRP‐conjugated anti‐GAPDH (Kangcheng, Shanghai, China) antibodies. The protein bands were analyzed using Image Lab analysis (Bio‐Rad).
The TNF‐α, IL‐1β, and IGF‐1 levels in the supernatants were detected using an ELISA according to the manufacturer's instructions (EK2822, EK201B2, Multi Sciences, Hangzhou, China; MG100, R&D). The sample concentrations were calculated using an equation generated from a standard curve.
The TNF‐α, IL‐1β, and IGF‐1 levels in the supernatants were detected using an ELISA according to the manufacturer's instructions (EK2822, EK201B2, Multi Sciences, Hangzhou, China; MG100, R&D). The sample concentrations were calculated using an equation generated from a standard curve.
8
Immunoprecipitation of Bad Protein
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Cells were lysed in NP-40 buffer (Beyotime) and centrifuged at 12000g for 10 min. The resulting supernatants were incubated with primary antibody of Bad (Santa Cruz) overnight at 4 °C. Subsequently, supernatants were incubated with Protein A/G PLUS-Agarose (Santa Cruz) for 2 h. Next, the resulting supernatants were washed with cold NP-40 buffer. The co-precipitated proteins were removed from the agarose beads by boiling in sodium dodecyl sulfate (SDS) sample buffer.
9
Western Blot Analysis of Metalloproteinases
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The tissue and cell samples were lysed using NP-40 buffer (Beyotime), and proteins were degenerated by heating. Then, an aliquot of 20 μg protein samples were electrophoresed on 10% bis–tris-acrylamide gel (Thermo Fisher), followed by the wet-transfer with the nitrocellulose membranes (Pall Life Science, Beijing, China). After blocking in skimmed milk (Solarbio), the membranes were probed with anti-matrix metalloprotein 9 (anti-MMP9; 1:1000; Affinity, Nanjing, China), anti-MMP2 (1:1000; Affinity), anti-MAP3K3 (1:1000; Affinity) and anti-GAPDH (1:8000; Affinity). After that, the secondary antibody against goat anti-rabbit IgG (1:5000; Affinity) was incubated with the membranes. The protein signals were detected with eyoECL Plus (Beyotime). GAPDH was employed to normalize protein abundance.
10
Western Blotting Analysis of Inflammatory Markers
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Kidneys were homogenized and lysed in NP-40 buffer (Beyotime Institute of Biotechnology, Haimen, China). Following 5–10 min boiling, homogenates were centrifuged at 10,000 × g at 4°C for 10 min to obtain the supernatant. Protein samples (50 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (w/v) Tween 20 for 2 h at room temperature, and subsequently incubated with the following primary antibodies: TNF-α (cat. no. sc-52746), IL-1β (cat. no. sc-515598), IL-6 (cat. no. sc-32296), NF-κB (cat. no. sc-114) and β-actin (cat. no. sc-517582; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA; all 1: 1,000), at 4°C overnight. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti IgG (cat. no. sc-516102; 1:10,000; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Signal detection was carried out with an enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA), and protein bands were analyzed with Quantity One® software version 4.5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blotting, n=8 in the control group; n=6 in CLP group (2 mice succumbed within 24 h); n=7 in UA(L)+CLP group (1 mouse succumbed within 24 h); n=8 in UA(H)+CLP group.
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