Western blotting experiments were performed using SDS-PAGE electrophoresis for total protein fractions of mouse liver samples as described elsewhere32 (link). Primary antibodies used for detection were anti-PXR (PA5-41170) and anti-CYP3A11 (PA1-343, Thermo Fisher Scientific). Protein band intensity on PVDF membranes was quantified using Quantity One imaging software (Bio-Rad, Hercules, CA, USA). Protein expression was normalized to expression of β-actin (A5316, Sigma–Aldrich). The results are presented as relative change in protein expression compared to corresponding controls in the same interval after PCN application. The data are shown as the mean ± SD acquired from five biological samples per group. For Western blotting experiments with primary human hepatocytes and cancer cell lines lysates, β-actin (A5316, Sigma–Aldrich), PXR (PA5-41170, Thermo Fisher Scientific), CYP3A4 (PA1-343, Thermo Fisher Scientific), and RXRα (5388, Cell Signaling Technology, Danvers, MA, USA) primary antibodies were used.
A5316
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada, Germany, Japan
The A5316 is a specialized laboratory equipment designed for precise and accurate measurements. It features advanced technology to ensure reliable and consistent performance. The core function of this product is to provide precise data and analytical capabilities to support scientific research and investigations. Further details about its intended use or specific applications are not available.
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Lab products found in correlation
β actin, by Merck Group (10 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Anti β actin, by Merck Group (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Ab1791, by Abcam (6 mentions)
Mammalian cell lysis kit, by Merck Group (4 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (4 mentions)
F7425, by Merck Group (4 mentions)
Mouse anti β actin, by Merck Group (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Thermo Fisher Scientific (4 mentions)
Supersignal west dura extended duration, by Thermo Fisher Scientific (3 mentions)
Pico plus chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Ab16502, by Abcam (3 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
203 protocols using a5316
1
Quantitative Western Blotting of PXR and CYP3A
2
Western Blot Analysis of GR Protein
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Heart tissue was extracted for protein using RIPA lysis buffer (Life Technologies, Carlsbad, CA, United States) and proteinase inhibitors cocktail (Pierce Biotechnology, Rockford, IL, United States). Protein concentrations were measured using the BCA kit (Pierce). Samples with equal amounts of proteins were separated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore Corporation, Billerica, MA, United States). After blocking with 5% non-fat milk in TBS (blocking buffer) for 2 h at room temperature, membranes were then incubated with primary antibodies against GR (1:1000; sc-1002; Santa Cruz Biotechnology, Santa Cruz, CA, United States) or β-actin (1:6000; A5316; Sigma-Aldrich, St. Louis, MO, United States) in blocking buffer at 4°C overnight followed by secondary antibody (1:4000; Santa Cruz Biotechnology) for 1 h at room temperature. The bands were visualized using Amersham ECLTM Western Blotting Detection Reagents (GE Healthcare, United States), and the blots were exposed to Hyperfilm. The results were analyzed using the Kodak ID image analysis software. GR protein abundance was normalized to β-actin.
3
GABA Receptor Expression in Chick Telencephalon
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An immunoblot analysis was performed as described previously (Yamaguchi et al., 2007 (link)). In brief, the telencephalons from 0- and 5-day-old chicks reared in the dark were dissected after anesthesia. For the detection of GABA-A receptors, an anti-GABA-A receptor subunit alpha 1 rabbit polyclonal antibody was used as the primary antibody (ab33299, 1:1,500; Abcam plc, Cambridge, United Kingdom), while an anti-rabbit horseradish peroxidase-conjugated antibody (1:1,000; GE Healthcare, Chicago, IL, United States) was used as the secondary antibody. To detect GABA-B receptors, an anti-GABA-B receptor subunit 2 rabbit monoclonal antibody (ab75838, 1:1,500; Abcam plc) was used, while an anti-rabbit horseradish peroxidase-conjugated antibody (1:1,000, GE Healthcare) was used as the secondary antibody. Data of each sample were normalized to the expression of beta-actin as detected by an anti-beta-actin mouse monoclonal antibody (A5316, 1:1,000, Sigma-Aldrich Co.). The band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, United States), and the ratios of the band intensities were calculated.
4
Mammalian Cell Lysis and Western Blot Analysis
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Treated cells or isolated MIVs were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich), as described previously (Niu et al., 2014 (link)). Equal amounts of protein were electrophoresed in a SDS-polyacrylamide gel under reducing conditions followed by transfer to PVDF membranes. Blots were blocked with 5% BSA in TBS-Tween, and Western blots were probed with antibodies specific for σ-1R (15168-I-AP; Proteintech Group), Src (2108; Cell Signaling), p-PDGFR-β (3161; Cell Signaling), NG2 antibodies (ab129051; Abcam), TBX18 (ab115262; Abcam), αSM (A5228; Sigma-Aldrich), and histone H3 (9715; Cell Signaling) at 1:1,000 dilution; NF-κB (ab16502; Abcam) at 1:2,000 dilution; ganglioside GM1 (bs-2367R; One World Lab) at 1:500 dilution; and p-Src (ab32078; Abcam), PDGFR-β (ab32570; Abcam), Desmin (ab32362; Abcam), and β-actin (A5316; Sigma-Aldrich) at 1:5,000 dilution. Secondary antibodies were alkaline phosphatase conjugated to goat anti-mouse/rabbit IgG, or rabbit anti-goat IgG (1:10,000; Jackson ImmunoResearch Labs). Signals were detected by SuperSignal West Dura Extended Duration or Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). All experiments had at least four biological replicates, and representative blots are presented in the figures.
5
Immunoblotting Analysis of Sigma-1 Receptor in Tumor Samples
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Tumor samples were processed as described earlier [54 (link)]. Preparation of cell lysates, SDS-PAGE and immunoblotting were performed as described elsewhere [55 (link)]. In brief, 40–80 µg protein per lane was transferred to PVDF membranes using a semi-dry transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked for 60–90 min with non-fat dry milk powder (5%, w/v) in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBS-T). For detection of Sig1R, membranes were incubated with primary antibodies in bovine serum albumin (BSA, 2% w/v) in TBS-T (PA5-30372, 1:500, Thermo Fisher Scientific (Waltham, MA, USA), (tumor samples), respectively, ab53852, 1:200, Abcam (Cambridge, UK) (cell lysates)), followed by incubation with peroxidase-conjugated secondary antibody (anti-rabbit IgG, A0545, 1:5000, Sigma-Aldrich, Steinheim, Germany). Proteins were visualized using Super Signal West Pico/Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and a CELVIN®S Chemiluminescence Imaging system (Biostep, Burkhardtsdorf, Germany). For detection of loading control, membranes were stripped and further processed using mouse anti-β-actin antibody (A5316, 1:1000, Sigma-Aldrich) and anti-mouse IgG (A9044, 1:10,000, Sigma-Aldrich) as described elsewhere [54 (link)].
6
Western Blotting of 5-HT6 Receptor
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Rats went through procedures in Figure 1 without cannula placement, microinjection or locomotion test. Western blotting was performed as previously described (Zhang et al., 2018 (link)). Briefly, brain tissue containing the VLO was rapidly collected after animal anesthesia and decapitation. The tissue was dissected and homogenized in ice-cold lysis buffer. The homogenate was incubated on ice for 30 min and then centrifuged at 10,000 g for 15 min at 4°C. The supernatant was collected and stored at −80°C until use. Sample protein levels were measured using a bicinchoninic acid (BCA) protein assay. Loading proteins were separated on acrylamide gels and then transferred to pore size 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat milk, the membranes were probed for anti-rabbit 5-HT6 receptor (Abcam, ab103016, 1:1,500, Cambridge, MA, United States), and anti-mouse actin (Sigma, A5316, 1:100,000, St Louis, MO, United States). Densitometry of each protein was evaluated by Image-J (NIH). Samples from each animal were run at least four times to minimize inter-blot variance. Raw values were normalized to actin.
7
Protein Extraction and Western Blot Analysis
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For protein isolation from cells, RIPA buffer with proteinase inhibitor cocktail was used. The protein concentration was measured by BCA assay. Standard Western blot techniques and a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA) were used according to previously described procedures [37 (link)]. The primary antibodies used were as follows: Gabra3 (1:1000, ab72446, Abcam, Cambridge, UK), p-p70s6k (1:1000, 9204, CST, Danvers, MA, USA), p70s6k (1:1000, 2708, CST), p-mTOR-S2448 (1:1000, 5536, CST), mTOR (1:1000, 2972, CST), p-AKT-S473 (1:2000, 4060, CST), AKT (1:2000, 2920, CST), MMP-9 (1:1000, 13,667, CST), MMP-2 (1:1000, 87,809, CST), p-c-Jun (1:1000, 9164, CST), c-Jun (1:1000, 2315, CST), p-JNK1/2 (1:1000, 4668, CST), JNK1/2 (1:1000, ab4821, Abcam), and β-actin (1:2000, A5316, Sigma, St. Louis, MO, USA).
8
Protein Extraction and Immunoblotting for USP21 in Cell Lysates
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Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; EMD Millipore) as previously described (22 (link)–24 (link)). Cells were centrifuged at 4°C for 10 min at 16,000 × g. Protein concentrations were determined by the Bradford assay (25 (link)). Aliquots containing 20 µg of total protein were separated by 10% sodium dodecyl-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:5,000, mouse monoclonal; A5316; Sigma-Aldrich; EMD Millipore). Appropriate secondary antibodies (1:3,000; rabbit anti-goat; ab6741; 1:5,000; rabbit anti-mouse; ab97046; Abcam, Cambridge, MA, USA) conjugated to horseradish peroxidase and enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) were used to detect the bound primary antibodies. Co-IP was performed with cell lysate (500 µg) incubated with USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc.), relA (1:1,000; rabbit polyclonal; sc-372; Santa Cruz Biotechnology, Inc.) or non-specific-IgG antibodies (1:1,000; rabbit IgG, monoclonal; ab172730; Abcam) using µMACS™ Protein A/G MicroBeads and MACS® Separation Columns according to the manufacturer's protocol (Miltenyi Biotec, Auburn, USA).
9
Inflammasome Activation Signaling Pathway
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Ultrapure lipopolysaccharide (LPS) (tlrl-3pelps), flagellin (Salmonella typhimurium) (tlrl-stfla), and MDP (tlrl-mdp) were from Invivogen (San Diego, CA, USA). The poly(dA:dT) (P0883) and ATP (A2383) were from Sigma-Aldrich (St. Louis, MO, USA). The Z-VAD-FMK (2163) was from Tocris Bioscience (Minneapolis, MN, USA). The following antibodies were used: polyclonal rabbit anti-caspase-1 for mouse caspase-1 (1:1000) (SC-514, Santa Cruz Biotechnology, Dallas, TX, USA); polyclonal goat anti-IL-1β for mouse IL-1β (1:1000) (AF-401-NA, R&D Systems, Minneapolis, MN, USA); polyclonal rabbit anti-ASC for mouse ASC (1:1000) (ADI-905-173-100, Enzo Life Sciences, Farmingdale, NY, USA); and monoclonal mouse anti-β-actin (1:5000) (A5316, Sigma-Aldrich).
10
Western Blot Antibody Panel
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We used the following antibodies in western blot analyses: mouse anti-Bcl-2 (B3170, Sigma), mouse anti-Bax (B8429, Sigma), mouse anti-RAD51 (SAB1406364, Sigma), mouse anti-β-actin (A5316, Sigma), and lgG-Peroxidase rabbit anti-mouse (A9044, Sigma).
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