Protein samples from the biological replicates used for phosphoproteomic analysis were lysed in 8 M urea as described above. Th17 cells were lysed at 20 × 106/mL in RIPA buffer (100 mM HEPES [pH 7.4], 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycho- late, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP (Pierce), and protease and phosphatase inhibitors [Roche]). Protein samples were mixed with NuPAGE LDS sample buffer (Life Technologies) supplemented with TCEP as reducing agent (25 mM; Sigma-Aldrich). Protein lysates were separated by SDS-PAGE polyacrylamide gel electrophoresis and then transferred to nitrocellulose membrane (Amersham) using standard conditions (Mini-PROTEAN tetra cell system; Bio-Rad). Membranes were blocked with 5% (w/v) nonfat dried skimmed milk powder in phosphate-buffered saline (PBS) containing 0.2% Tween 20. Membranes were probed with the following primary antibodies: pSTAT3-Y705, pSTAT3-S727, STAT3, RLC (all purchased from Cell Signaling Technologies), pRLC-S19/S20 (Rockland Inc.), SCM1 (Bethyl Laboratories) following manufacturer´s recommendations. Primary antibodies were detected using peroxidase-conjugated secondary antibodies (Goat anti-Rabbit-Ig and Goat anti-Mouse-Ig, Thermo Scientific), and chemiluminescence detected with the ImageQuant LAS-4000 imaging system (Fujifilm).
Imagequant las 4000 imaging system
Manufactured by Fujifilm
Sourced in United States, Japan
The ImageQuant LAS-4000 is a high-performance imaging system designed for life science applications. It captures and analyzes images of samples, such as gels, blots, and arrays, using sensitive detection technologies. The system is capable of detecting a wide range of signals, including chemiluminescence, fluorescence, and bioluminescence.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
P stat3 s727, by Cell Signaling Technology (1 mentions)
Goat anti rabbit ig, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse ig, by Thermo Fisher Scientific (1 mentions)
P stat3 y705, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Mini protean tetra cell system, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Proteoguard edta free protease inhibitor cocktail, by Takara Bio (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Image gauge analysis software, by Fujifilm (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime western blotting detection reagent, by Cytiva (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Can get signal solution 1, by Toyobo (1 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
5 protocols using imagequant las 4000 imaging system
1
Immunoblotting of Phosphorylated Proteins
2
Western Blot Analysis of Protein Expression
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Cells were lysed by incubation for 20 min at 4 °C in TBS-lysis buffer (comprising TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) supplemented with 1% (v/v) Triton X-100 and 1× ProteoGuard EDTA-Free Protease Inhibitor Cocktail (Takara Bio, Kusatsu, Japan)). The supernatant was collected after centrifuging the sample at 15,000 rcf for 20 min at 4 °C and was then boiled in Laemmli buffer before separation by SDS-PAGE and transferal to a PVDF membrane. Membranes were incubated for 1 h at room temperature with blocking solution (TBS containing 0.1% Tween-20 and 5% nonfat milk) and then overnight at 4 °C with the appropriate primary antibody. Subsequently, the membranes were washed with TBS and incubated for 1 h at room temperature with the secondary antibody (HRP-conjugated goat antirabbit or goat antimouse antibody) (Bio-Rad, Hercules, CA, USA). Protein bands were visualized and quantified using Clarity Western ECL Substrate (Bio-Rad) and the ImageQuant LAS 4000 Imaging System (Fujifilm).
3
Protein Concentration Quantification in Hippocampal Tissue
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Protein concentration was assayed after extraction of bilateral hippocampal tissue protein supernatant with protein lysate. The sample volume for the initial determination of the internal reference was 6 μL per well, the electrophoresis current was kept as constant as possible, and the electrophoresis voltages and time were as follows: 20 V-10 min, 60 V-20 min, and 100 V-60 min, respectively. The polyvinylidene fluoride (PVDF) membrane was removed and placed in triethanolamine-buffered saline (TBS) with Tween 20 (TBS-T) for washing (5 min × 3 times) and was blocked at room temperature (23-26°C) for 1 h. After these steps, the membrane was incubated with primary at 4°C overnight. Then, the membrane was washed and incubated in secondary antibody for 1 hours at room temperature for 1 h. Immunoreactive bands were visualised using the Image Quant LAS 4000 imaging system (Fujifilm USA, Valhalla, NY, USA), and densitometric analyses were performed using Image Gauge analysis software (Fujifilm, USA). The brightness and contrast of the raw blots were equally adjusted across the entire image using Adobe Photoshop CS5 software (Adobe Systems, Ottawa, ON, Canada) to generate representative images [34 (link)].
4
Quantitative Western Blot Analysis
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Western blotting was performed according to the standard procedures [49 (link)]. RIPA buffer mixed with protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) was used to prepare protein extracts. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Synaptosomal extracts were separated by polyacrylamide gel electrophoresis (4–12% SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with TBS-T containing 5% BSA. The following primary antibodies were used: rabbit polyclonal antibodies directed against phospho-PKC-α, phospho-SNAP-25 and β-actin (Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibodies (BioLegend, Inc., San Diego, CA, USA) were used to detect the respective primary antibodies. Immunoreactive proteins were developed with Amersham ECL prime Western blotting detection reagent (Cytiva, Marlborough, MA, USA). Chemiluminescent signals were detected with an ImageQuant™ LAS-4000 imaging system (Fujifilm, Tokyo, Japan). The level of expression or phosphorylation was quantified using Multi Gauge V3.0 (Fuji Photo Film. Co. Ltd., Mini-ku, Tokyo, Japan).
5
Western Blot Analysis of siRNA-Transfected OPCs
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siRNA-transfected OPC cells were lysed with RIPA Buffer (Cell Signaling Technology). The lysates were quantified by DC Protein Assay Kit II (BIO-RAD), and then 5 μg of the lysates separated by electrophoresis in a 7.5–15% polyacrylamide gel (DRC) and blotted onto a PVDF membrane (Millipore). The membrane was blocked with 3% BSA in TBS, incubated with primary antibody diluted in Can Get Signal Solution I (TOYOBO) overnight, incubated with HRP-linked anti-mouse or anti-rabbit IgG diluted in TBS-T for 1 h, and developed with ECL (GE Healthcare). Images were visualized using an ImageQuant LAS-4000 imaging system (Fujifilm) or iBright Imaging Systems (Thermo Fisher Scientific).
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