Cultured C2C12 cells were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Wako) with protease and phosphatase inhibitors (Nacalai Tesque Inc.) and processed for western blotting analyses as previously described [25 (link),26 (link)]. Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA). Luminescence signals from ECL reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) were detected using LAS-4000 (Fujifilm Corp., Tokyo, Japan). Quantitative densitometric analyses were performed using Fiji software.
Il 4rα
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
IL-4Rα is a protein that functions as the alpha subunit of the interleukin-4 receptor. It is responsible for binding interleukin-4, a cytokine involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Myomaker, by Abcam (1 mentions)
Protease and phosphatase inhibitors, by Nacalai Tesque (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
P stat6, by Santa Cruz Biotechnology (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
P jak1, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Santa Cruz Biotechnology (1 mentions)
4 protocols using il 4rα
1
Protein Expression Analysis in C2C12 Cells
2
Protein Expression Analysis via Western Blot
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Western blot analysis was performed according to previously published procedures [36 (link)]. Tissue samples were harvested with RIPA lysis buffer containing 1% proteinase inhibitor cocktail (Sigma-Aldrich) according to protocols, and then centrifuged to collect supernatants. SDS Loading Buffer was added to supernatants, and then boiled before separated by 10% SDS-PAGE. After electrophoretically transferring protein to polyvinylidene difluoride membranes (Millipore), the membranes were incubated with primary antibodies, ITGB4 Abcam ab182120, 1:1000, synaptophysin (SYP) Millipore MAB5258-I, 1:1000, TNFRα Santa Cruz sc-8436, 1: 1000, IBA1 Santa Cruz sc-32,725, 1: 1000, and IL-4Rα Santa Cruz sc-28361, 1: 1000, and subsequently reacted with horseradish peroxidase-conjugated secondary antibody prior to visualizing through the use of ECL reagents (Pierce).
3
Western Blot Analysis of Apoptosis and Cytokine Signaling
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B16F10-bearing melanoma tumor tissues were homogenized with a protein extraction solution (PRO-PREP, Intron Biotechnology) and lysed by 60-minute incubation on ice. The cell lysate was centrifuged at 15,000 rpm for 15 minutes at 4°C. Equal amounts of protein (20 μg) were separated on an SDS/12% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (GE Water and Process Technologies, Trevose, PA). Blots were blocked for 1 hour at room temperature with 5% (w/v) skim milk in TBS with Tween-20 (TBST: 10 mM Tris, pH 8.0, and 150 mM NaCl solution containing 0.05% Tween-20). After a brief wash in TBST, the membranes were immunoblotted with primary antibodies targeting the following proteins: caspase-3, caspase-9, caspase-8, BCL-2 (1:1000 dilutions; Cell Signaling Technologies, Beverly, MA), BAX, IL-4Rα, JAK1, p-JAK1, STAT6, p-STAT6 (1:1000 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), and IL-4 (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA). The blots were performed using specific antibodies followed by secondary antibodies and visualized on an enhanced chemiluminescence detection system.
4
Silencing IL4Rα and IL13Rα1 in SNU308 Cells
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SNU308 cells were plated in 60 mm dishes (3 × 105 cells/dish) and incubated overnight. Then, 0.6 mL of warm serum free media was added to a 1.7 mL tube. 5 uL of control, IL4Rα, or IL13Rα1 siRNA (Santa Cruz, the concentration of siRNA stock solution is 10 uM) and 15 µL of lipofectamine 2000 (Waltham, MA, USA) were added to media respectively and incubated for 15 min. After incubation, the media containing siRNA and the media containing lipofectamine 2000 were mixed and incubated for 15 min. After 15 min of incubation, cells were washed twice with PBS and the siRNA and lipofectamine 2000 mixtures were added to cells. Cells were incubated at 37 °C in incubator for 6 h. After 6 h of incubation, the media was removed and fresh media containing FBS and antibiotics was added to cells.
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