Immunoblots were performed according to standard procedures 19 (link). The antibodies used for this study are following; The antibodies against AKT (2920S), p-AKT (4060S), YAP (14074S), p-YAP (13008), TAZ (83669S), p-TAZ (59971S), p65 (6956S), p-p65 (3033S) are obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-MRCKα (sc-374568) and anti-MRCKβ (sc-374597) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody for beta-actin (MA1-91399) was purchased from ThermoFisher Scientific. Anti-tubulin antibody (T5168) was purchased form Sigma-Aldrich (St. Louis, MO, USA). The chemiluminescent signals were detected by using iBright Imaging System (ThermoFisher Scientific). Signal intensity was measured using ImageJ software (https://imagej.nih.gov/ij/index.html ). Ponceau S staining was used to confirm that the samples were equally loaded.
Anti tubulin antibody t5168
Manufactured by Merck Group
Sourced in United States
The Anti-tubulin antibody (T5168) is a primary antibody used in immunological techniques to detect and study tubulin, a key structural protein found in the cytoskeleton of eukaryotic cells. This antibody is produced in mice and purified from the ascites fluid.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit 31460, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Anti mouse 115 035 003, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
X gal, by Promega (1 mentions)
Irdye800 conjugated anti rabbit igg 926 32211, by LI COR (1 mentions)
Endothelial cell growth supplement pack, by PromoCell (1 mentions)
Secondary peroxidase labeled anti mouse or anti rabbit immunoglobulin g antibodies, by Jackson ImmunoResearch (1 mentions)
Il 1β 3a6, by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
5 protocols using anti tubulin antibody t5168
1
Immunoblot Analysis of Cellular Signaling
2
Amplification and Cloning of tRNA Genes
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We performed PCR to amplify the genomic DNA fragments containing the different representative tRNAs using primer pairs (Table S1 and S2) and genomic DNA purified from HEK 293T as templates. The amplified PCR fragments were cloned into pGEM-T Easy vector (Promega, USA), according to the manufacturer's manual. A tRNAHisGUG gene, which was presented in the old tRNA database (http://gtrnadb2009.ucsc.edu ) but was removed out in the new database (http://gtrnadb.ucsc.edu ), was designated as a pseudo gene.
Primary antibodies were purchased from Cell Signaling Technology (USA). The antibodies used were phosphor (P)-p70 S6K (#9206), S6K (#9202), P-p90-RSK (#9341), P-MSK (#9591), RSK1/RSK2/RSK3 (#9355), MSK2 (#3679), P-ERK1/2 (#9101), and P-SAPK/JNK (#4668). Anti-tubulin antibody (T-5168) was purchased from Sigma-Aldrich (USA). Secondary antibodies, goat anti-rabbit (31460) and goat anti-mouse (Cat. 31430), were purchased from Thermo Fisher Scientific (USA).
Primary antibodies were purchased from Cell Signaling Technology (USA). The antibodies used were phosphor (P)-p70 S6K (#9206), S6K (#9202), P-p90-RSK (#9341), P-MSK (#9591), RSK1/RSK2/RSK3 (#9355), MSK2 (#3679), P-ERK1/2 (#9101), and P-SAPK/JNK (#4668). Anti-tubulin antibody (T-5168) was purchased from Sigma-Aldrich (USA). Secondary antibodies, goat anti-rabbit (31460) and goat anti-mouse (Cat. 31430), were purchased from Thermo Fisher Scientific (USA).
3
Protein Extraction and Immunoblotting Protocol
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Proteins were extracted using a lysis buffer containing 12mM Hepes, 300mM mannitol, 1mM EGTA, 1mM EDTA, 1%Triton X-100, and 0.1% SDS adjusted to pH 7.6, in the presence of 1mM n-ethylmaleimide, 1mM phenylmethanesulfonyl fluoride and protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein extracts were clarified by centrifugation and then quantified using the Pierce BCA Protein Assay kit (ThermoFisher). 20µg of protein extracts were subjected to SDS-PAGE and then transferred onto a PVDF membrane. Immunoblotting was carried out using either anti-CaSR antibody (ImmunoGenes) or anti-Tubulin antibody (T5168, Sigma-Aldrich). HRP-conjugated antirabbit (111-035-003, Jackson ImmunoResearch, USA) and anti-mouse (115-035-003, Jackson ImmunoResearch) were used as secondary antibodies. Chemiluminescence was detected by a ChemiDoc MP Imaging System (Biorad, USA).
4
Immunoblotting and Cell Signaling Assays
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All chemicals including those used for immunoblotting and anti-tubulin (T5168) antibody were obtained from Sigma (Buchs, Switzerland). Antibody against p21Cip1 (OP64) was purchased from Calbiochem (Genève, Switzerland); antibody against phosphor-p53-S15 (#9284s) was from Cell Signalling (Allschwil, Switzerland); antibodies against Arg-I (sc-18351), Arg-II (sc-20151) and p53 (sc-6243) were from Santa-Cruz (Nunningen, Switzerland); Alexa Fluor680-conjugated anti-mouse IgG (A21057); Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and dihydroethidium (DHE) were from Molecular Probes/Invitrogen (Lucerne, Switzerland); IRDye800-conjugated anti-rabbit IgG (926-32211) were from LI-COR Biosciences (Bad Homburg, Germany); the membrane-permeable 4,5-Diaminofluorescein diacetate (DAF-2DA) was from VWR international SA (Dietikon, Switzerland); X-gal was from Promega (Dübendorf, Switzerland); Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH (Allschwil, Switzerland) and all cell culture media and materials were purchased from Gibco BRL (Lucerne, Switzerland).
5
Western Blot Analysis of NLRP3 Inflammasome
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Cells were harvested, and culture supernatants were concentrated 30-fold using Amicon filter. Equal amounts of proteins were electrophoresed on Mini-PROTEAN® TGX™ gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes as described [45 (link)]. Antibodies to NLRP3 (D2P5E), caspase-1 (D7F10) and IL-1β (3A6) were purchased from Cell Signaling Technology; antibodies to ASC (F-9) and c-Myc (9E10) were purchased from Santa Cruz Biotechnology; an anti-Tubulin (T5168) antibody was purchased from Sigma-Aldrich. Secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibodies were purchased from Jackson ImmunoResearch Laboratories, West Grove, PA, USA.
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