Cells and tissues were lysed by RIPA buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes (Pierce, Rockford, IL, USA). The membranes were blocked with 5% nonfat dry milk in TBST. Primary antibodies were: SMAD7 (MAB2029, R&D Systems, Minneapolis, MN, USA), P21 (SAB4500065, Sigma Aldrich), pSMAD2 (Ser465/467), pSMAD1/3, BCL-2, BCL-XL, pSTAT3, pJNK, pc-JUN, MCL-1, cleaved caspase-3 and Actin (3101, 9520,2870, 2762, 9145, 4668, 3270,5453,9661 and 3700, Cell Signaling Technology), c-MYC, pIκBα, IL-6, TGF-β1, pERK, CyclinD1, pP38 and VEGF (sc-40, sc-1265, sc-8404, sc-130348, sc-13073, sc-753, sc-7973, and sc-507, Santa Cruz Biotechnology). Secondary antibodies were: horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies (Santa Cruz Biotechnology). The membranes were developed using Supersignal Ultra (Pierce).
Sc 8404
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-8404 is a general-purpose laboratory centrifuge designed for a wide range of applications. It features a compact and durable construction, enabling efficient separation of samples in various fields of research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab6671, by Abcam (3 mentions)
Ab205718, by Abcam (2 mentions)
Ab288751, by Abcam (2 mentions)
Ab280209, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
P smad2, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Smad7, by R&D Systems (1 mentions)
Sc 1265, by Santa Cruz Biotechnology (1 mentions)
Sc 13073, by Santa Cruz Biotechnology (1 mentions)
Sc 753, by Santa Cruz Biotechnology (1 mentions)
Sc 7973, by Santa Cruz Biotechnology (1 mentions)
Sc 507, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Sc 40, by Santa Cruz Biotechnology (1 mentions)
Tgf β1, by Santa Cruz Biotechnology (1 mentions)
11 protocols using sc 8404
1
Protein expression analysis by Western blot
2
Protein Expression Analysis in Aortic Tissue
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The aortas were washed three times in TBS, and then lysed in RIPA buffer. The lysates were centrifuged at 14,000 × g for 30 min at 4°C. The total protein concentration of each sample was measured using a Micro BCA Protein Assay reagent kit (Pierce, Rockford, IL, USA). The same amount of lysate from each line in SDS sample buffer was resolved by 10% SDS-polyacrylamide gel electrophoresis and electroblotted onto a PVDF membrane, which was then blocked with 5% fat-free milk in TBST (TBS, 0.1% Tween-20) for 1 h at room temperature. The ET-1 Ab (1:250 dilution; sc-21625-R; Santa Cruz Biotechnology, Inc.); phosphorylated (p-)AMP-activated protein kinase (AMPK) (Thr172) Ab (1:500 dilution; no. 2535) and p-nuclear factor (NF)-κB p65 Ab (1:500 dilution; no. 3033) (both from Cell Signaling Technology, Inc., Danvers, MA, USA); and p-IκBα Ab (1:250 dilution; sc-8404; Santa Cruz Biotechnology, Inc.) were incubated overnight at 4°C, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). Specific bands of the target proteins were stained with chemiluminescence reagent (Pierce). Densitometric scanning of the exposed X-ray film was used for semi-quantitative measurement of the protein bands. β-actin levels were used to normalise the relative protein expression levels.
3
Immunoblotting Analysis of Apoptosis and Stress Markers
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Total protein was extracted from cells or lung tissues using RIPA lysis, and concentration was determined via BCA assay kit. Equal amounts of protein were then separated via SDS-PAGE and transferred to a PVDF membrane. Blots were incubated with primary antibodies overnight. The primary antibodies are as follows: anti-Cleaved Caspase-3 (1:500; ab2302; Abcam), anti-Bax (1:500; ab32503; Abcam), anti-Bcl-2 (1:500; ab182858; Abcam), anti-β actin (1:500; ab8226; Abcam), anti-Nrf2 (1:500; ab137550; Abcam), anti-Histone H3 (1:500; ab1791; Abcam), p-IκBα (1:500; sc-8404 Santa Cruze), IκBα (1:500; ab32518; Abcam), and NF-κB (1:500; 10,745-1-AP; Proteintech). HRP-conjugated goat anti-rabbit IgG (1:1000; 7074; Cell signaling) was used as secondary antibody. β-actin and Histone H3 was used as the internal reference and quantification of the band densities was obtained using the ImageJ software.
4
Western Blot Analysis of Protein Expression
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Expression of proteins was examined by western blotting. The standard procedures including SDS-polyacrylamide gel electrophoresis, membrane transference, antibodies incubation, and enhanced chemiluminescence detection were applied for 20 μg of total protein. The primary antibodies including CCL16 (ab199162, 1:2,000), B cell lymphoma (Bcl)-2 (ab196495, 1:2,000), Bcl-2-associated X protein (Bax; ab199677, 1:1,000), cleaved-caspase-3 (ab49822, 1:500), IL-6 (ab208113, 1:1,000), IL-1β (ab2105; 1:1,000), TNF-α (ab6671, 1:2,000), and GAPDH (ab8245, 1:10,000) were provided by Abcam (Cambridge, UK). Primary antibodies against TLR4 (sc-293072, 1:500) and phosphorylated P65 (p-P65; sc-136548, 1:500) and p-IκB-α (sc-8404, 1:500) were obtained from Santa Cruz (Shanghai, China). The polyvinylidene fluoride (Millipore, Billerica, MA, USA) membrane used for protein transfer was blocked with 5% non-fat milk at room temperature for 1 h. Then, the membranes were incubated with the aforementioned primary antibodies at 4°C overnight and reincubated with horseradish peroxidase-labeled secondary antibodies against Rabbit IgG (ab205718, 1:50,000; Abcam) and Mouse IgG (ab97023, 1:20,000; Abcam) at room temperature for 2 h. The relative protein levels were normalized to GAPDH and then compared to control.
5
Immunofluorescence Analysis of NF-κB Pathway
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Paraffin sections were dewaxed and repaired with high pressure antigen, and then 3% hydrogen peroxide was added to remove endogenous enzymes. After blocking with normal fetal bovine serum, NF-κB p65 (ab288751, Abcam), p-IκBα (sc-8404, Santa Cruz Biotechnology), and TOPK (ab280209, Abcam) antibodies were incubated overnight at 4°C. Anti-Human IgG1 H&L (Alexa Fluor® 488) (ab200622, Abcam) or Goat Anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11014, Invitrogen) were used as secondary antibodies. The nuclei were stained with DAPI (Invitrogen). The fluorescence was observed under IX73 inverted fluorescence microscope (Olympus Company, Japan).
6
Immunofluorescence Analysis of NF-κB Pathway
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The cells were first grown on an 8-well slide; then, they were incubated with the NucBlue® Live Cell Stain reagent (Invitrogen) for 20 min, fixed with 4% paraformaldehyde for 15 min, and incubated with 0.1% Triton 100 for 20 min. After being blocked with 2% BSA in PBS for 20 min, the cells were incubated with the following primary antibodies overnight at 4 °C: NF-κB antibodies (1:100, ab32536, Abcam), TNF-α antibodies (1:100, ab6671, Abcam), TAK1 antibodies (1:1000, ab109526, Abcam), TAB1 antibodies (1:200, ab227210, Abcam), and p-IκBα (B-9) antibodies (1:100, sc-8404, Santa Cruz Biotechnology). The cells were then washed with PBS, incubated with the corresponding secondary antibodies for 1 hour, and visualized using a fluorescent microscope (DM16000B, Leica, Heerbrugg, Switzerland). The tissues were cut into 8-μm-thick sections. After dewaxing, dehydration, and antigen retrieval, the sections were blocked using 5% serum, incubated with the primary antibodies overnight at 4 °C as mentioned above, and then incubated with biotin-labeled secondary antibodies for 1 h at room temperature.
7
Western Blot Analysis of Penumbra
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For WB analysis, we used peri-infarct (penumbra) cortical regions. Using a brain matrix, the brains were rapidly dissected into 4.0 mm coronal sections (approximately 0.5 mm and −3.5 mm from bregma). Brain tissue was homogenized and processed for western blotting as previously described. Fifty-microgram of proteins were loaded in each lane and separated followed by transfer to nitrocellulose membranes. The membranes were blocked for non-specific binding and probed with primary antibodies against NLRP3, Caspase-1, ASC (1:1000; AG-20B-0014; AG-20B-0042; AG-25B-0006 Adipogen life sciences), cleaved IL-1β, Caspase-3, Phospho- NFκBp65, NFκBp65 (1:1000; CST-12242; 9664; 3033; 8242; Cell signaling technology), Cleaved PARP, Anti IL-18, IL-1β (1:1000; ab32064; ab71495; ab9722; Abcam, USA), phospho IκB (1:1000; Santa cruz biotechnology, SC8404) at 4 °C for overnight. Following TBS-T washes, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1: 10,000; Sigma). The bands were then visualized by means of an enhanced chemiluminescent substrate system (Thermo fisher scientific). Protein levels were analyzed densitometrically, using Image J software and were normalized to loading controls, and expressed as fold change.
8
Immunohistochemical Analysis of NF-κB, Autophagy, and Cell Signaling
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The tissue specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned continuously at 4 to 5 μm. After dewaxing and repairing with high-pressure antigen, samples were incubated with NF-κB p65 (ab288751, Abcam), Beclin-1 (ab207612, Abcam), TOPK (ab280209, Abcam), LC3 (ab128025, Abcam), and p-IκBα (sc-8404, Santa Cruz Biotechnology) at 4°C overnight. Subsequently, Rabbit Anti-Human IgG H&L antibody (ab6759, Abcam) was used as the secondary antibody. DAB staining, hematoxylin restaining, hydrochloric alcohol differentiation, and blue return were added. Finally, the sections were dehydrated, hyalinized, sealed, and microscopically examined. The target protein staining was in brown, and the nucleus was in blue-purple.
9
Western Blot Analysis of Inflammatory Proteins
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Western blotting for TNFαip2, TNF-α, β-Actin and p-IκB was performed as previously described.33 (link) Briefly, the concentration was measured with the Bradford method (Bio-Rad Protein Assay kit; Bio-Rad Laboratories, Hercules, CA, USA) and then run on 10% polyacrylamide gel. Before protein transfer, PVDF membranes were incubated with the primary antibodies overnight at 4 °C and then incubated with horseradish peroxide-conjugated anti-rabbit or anti-mouse secondary antibody at 37 °C for 1 h. Primary antibodies were: TNFαip2 (SC-28318, Santa Cruz Biotechnology, Inc.), p-IκB (sc-8404, Santa Cruz Biotechnology, Inc.), TNF α (ab6671, Abcam, Cambridge, MA, USA) and anti-β-Actin (ab8227, Abcam).
10
Inflammatory Pathway Antibody Validation
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Antibodies against AIM2 (ab180665), Caspase 1 (ab179515), NLRC4 (ab201792), and NLRP3 (ab214185) were purchased from Abcam (Cambridge, UK). Antibodies against p-IκB (Sc-8404), IKK (Sc-7607), and p-IKK (Sc-21660) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Antibodies against HIF-1α (14179s), IL-1β (12426s), mTOR (2972s), and p-mTOR (5536s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against IκB (51066-1-AP), p65 (10745-1-AP), and β-actin (60008-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). Antibody against NALP1(PA5-17275) was purchased from Thermo Fisher (Danvers, MA, USA). Antibody against p-P65 (bs-0982R) was purchased from Bioss (Beijing, China).
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