2-Methoxystypandrone was isolated from the root of P. cuspidatum Sieb, as reported previously;(21 (link)) DTT and MTT were purchased from Genebase (Shanghai, China); GSH was obtained from Shanghai Sibas Bioscience (Shanghai, China); IL-6 and IFN-α were from Peprotech (Saint Paul, MN, USA); TNF-α was from R&D Systems (Minneapolis, MN, USA); IFN-γ was obtained from Shanghai Clone (Shanghai, China); anti-phosphorylated STAT3 (Tyr705), anti-phophorylated JAK2 (Tyr1007/1008), anti-JAK2, anti-phophorylated JAK1 (Tyr1022/1023), anti-JAK1, anti-phophorylated TYK2(Tyr1054/1055), anti-TYK2, anti-phosphorylated IκB-α (Ser32/36), anti-IκB-α, anti-phosphorylated IKK-αβ (Ser176/180 for IKKα and Ser177/181 for IKKβ) and anti-IKK-α antibodies were obtained from Cell Signaling Technology (Boston, MA, USA); anti-STAT3, anti-GP130 and anti-α-Tubulin antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA); anti-β-Actin antibody was from Abmart (Shanghai, China); anti-GAPDH antibody was obtained from Kang Chen Bioscience (Shanghai, China); secondary HRP-conjugated antibodies were from Multi Sciences Biotech (Hangzhou, China); and the Apoptosis Detection Kit was from Shanghai MaiYueEr Bioscience (Shanghai, China).
Anti β actin antibody
Manufactured by Abmart
Sourced in United States, China
The Anti-β-actin antibody is a laboratory reagent used for the detection and quantification of the β-actin protein in various biological samples. β-actin is a ubiquitous cytoskeletal protein found in all eukaryotic cells and is commonly used as a control or reference protein in various biochemical and cellular analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ifn α, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti gp130, by Santa Cruz Biotechnology (1 mentions)
Anti phosphorylated ikk αβ, by Cell Signaling Technology (1 mentions)
Secondary hrp conjugated antibodies, by MultiSciences Biotech (1 mentions)
Anti stat3, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved parp 1 antibody, by Cell Signaling Technology (1 mentions)
Whole protein extraction kit, by Keygen Biotech (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti non phospho active β catenin, by Cell Signaling Technology (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
5 protocols using anti β actin antibody
1
2-Methoxystypandrone Isolation and Reagents
2
Western Blot Analysis of Apoptosis Markers
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The cells were washed twice with phosphate-buffered saline (PBS), and total protein was extracted with a Whole Protein Extraction Kit (Keygen, Nanjing, China). Protein samples were separated by SDS−PAGE and then transferred to PVDF membranes. Subsequently, the membranes were blocked in 1 × casein buffer for 1 h at room temperature and then incubated with anti-cleaved-parp1 antibody (Santa Cruz, CA, USA), anti-cleaved-caspase 3 antibody (Cell Signaling Technology, USA), anti-GAPDH antibody (Abmart, Shanghai, China) and anti-β-actin antibody (Abmart, Shanghai, China) at 4°C overnight. The membranes were washed three times with TBS-T, incubated with secondary antibodies (Abmart, Shanghai, China) for 1 h, and detected with a gel imaging analysis system (Sinsage, Beijing, China).
3
Western Blot Analysis of Cell Signaling
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For WB, cells were collected and lysed in RIPA buffer (Cell Signaling) with a complete protease inhibitor cocktail (Roche), phosphatase inhibitors (Roche), and PMSF (Sigma), then centrifuged at 12,000g for 10 min at 4 °C. Protein concentration was measured by Bio-Rad protein assay kit (Hercules, CA) according to the manufacturer’s instructions. Western blot assays were performed as previously described12 (link). The primary antibodies were obtained as follows: anti-caspase-3, anti-PCNA, anti-β-Catenin, anti- Non-phospho (Active) β-Catenin, and Exosomal Marker Antibody Sampler Kit antibody (Flotillin-1 and EpCAM) from Cell Signaling Technology, anti-β-actin antibody from Abmart.
4
Molecular Mechanisms of Inflammasome Activation
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The following primary antibodies were used for Western blot, co-IP, and immunofluorescence: anti-HAX-1 antibody (BD, cat: 610825), anti-NLRP3 antibody (CST, cat: 15101), anti-ASC antibody (CST, cat: 67824), anti-Caspase1 antibody (CST, cat: 24232), anti-Cleaved Caspase1 antibody (CST, cat: 89332), anti-GSDMD antibody (CST, cat:93709), anti-TMEM119 antibody (Abcam, cat: ab209064), anti-β-actin antibody (Abmart, cat: P30002S), Goat anti-rabbit antibody HRP linked (CST, cat: 7074), Goat anti-mouse antibody (CST, cat:7076). The co-IP assays were performed by using Pierce Crosslink Magnetic IP Kit (Thermo Scientific, Cat: 88805). The immunofluorescence assays were performed by using Opal 7-color manual IHC kit (PerkinElmer, NEL801001KT). The following recombinant proteins were used for BLI assay: recombinant NLRP3 (Abmart, cat: EHH22785), recombinant ASC (Proteintech, cat: Ag28424), recombinant HAX-1 (Proteintech, cat: Ag27244). NLRP3 selective inhibitor MCC950 was purchased from APExBIO company, cat: C3780.
5
Myc-tagged RBM24a Protein Expression
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The coding sequence (CDS) of rbm24a with or without the 8-bp deletion was inserted into modified pEGFP-N2 vector with EGFP-CDS replaced by Myc-CDS. HEK293T cells were transfected with expression vectors using LipoMax transfection reagents (Sudgen, Cat. No. 32012). 24 h after transfection, cells were lysed in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris at pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 × protease inhibitor cocktail (Roche). The supernatant was then collected after centrifugation and separated by polyacrylamide gel electrophoresis (PAGE), then transferred to PVDF membrane. After blocking in TBS (20 mM Tris–HCl, 150 mM NaCl) containing 5% non-fat dry milk and 0.05% Tween-20, the membrane was incubated with anti-Myc antibody (Abclonal, Cat. No. AE010) or anti β-Actin antibody (Abmart, Cat. No. P30002) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (Bio-Rad, Cat. No. 170-6515 and 170-6516) at room temperature for an hour. The signals were detected with the ECL system (Thermo Fisher Scientific).
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