The KFs with different treatment were harvested by centrifugation and washed with PBS. Cells were lysed in RIPA buffer containing protease inhibitors. The lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were incubated overnight at 4 °C with the primary antibodies after blocking with 5% nonfat milk. The anti-human E-cadherin, anti-human vimentin, anti-human PKM2, anti-human HIF-1α, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The blots were then incubated with the secondary antibody for 2 h at room temperature. Each experiment was repeated for 3 times. Specific proteins were visualized using the ECL system (GE Healthcare) and the FUJIFILM Luminescent Image Analyzer LAS3000 (Fuji Film).
Luminescent image analyzer las3000
Manufactured by Fujifilm
Sourced in Germany, United States
The FUJIFILM Luminescent Image Analyzer LAS3000 is a laboratory equipment used for the detection and analysis of luminescent signals. It is designed to capture and quantify various types of luminescent samples, such as those used in genomics, proteomics, and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Western blotting luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Np 40, by Thermo Fisher Scientific (2 mentions)
Scion image software, by Techcomp Instruments (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti human e cadherin, by Cell Signaling Technology (1 mentions)
Anti human vimentin, by Cell Signaling Technology (1 mentions)
Anti human hif 1α, by Cell Signaling Technology (1 mentions)
Anti human phospho p70s6k, by Cell Signaling Technology (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
P stat6, by Cell Signaling Technology (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
4 protocols using luminescent image analyzer las3000
1
Western Blot Analysis of EMT Markers
2
Protein Extraction and Western Blot Analysis
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Cellular proteins were prepared with lysis buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP‐40 (28324, ThermoFisher Scientific), 1 mmol/L dithiothreitol (DTT), 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3PO4 and protease inhibitor (11 697 498 001, Hoffmann‐La Roche [Basel, Switzerland])]. The proteins were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidene difluoride membrane (Millipore), followed by blocking with 5% skim milk (232100, BD Difco) in TRIS‐buffered saline‐Tween 20 (20605, ThermoScientific Fisher) (TBST). The membranes were then incubated with primary antibodies overnight at 4°C on a rocker. After three washes in TBST, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (7076, 7074, Cell Signaling) for 1 hour at room temperature. The peroxidase activity was visualized by enhanced chemiluminescence using Western Blotting Luminol Reagent (sc‐2048 Santa Cruz Biotechnology) and FUJIFILM Luminescent Image Analyzer LAS‐3000 (Fujifilm Life Science). Quantification of Western blot analysis was performed after retrieving the density of the bands using Scion Image software (Scion Corporation) after more than three independent sets of experiments.
3
Phosphorylation analysis of STAT transcription factors
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To analyse the phosphorylation status of AD-and psoriasis-relevant transcription factors STAT1, STAT3 and STAT6, NHEKs were pre-incubated for 1 h with tofacitinib (2 lM) and stimulated with rhIL-4 (50 ng/mL) and rhIL-13 (50 ng/mL) or rhIL-17A (10 ng/mL), rhIL-22 (25 ng/mL) and rhTNFa (10 ng/mL) for 15 min. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific), supplemented with protease and phosphatase inhibitors, prior to mechanical disruption by sonification (S-250 D; Branson Ultrasonics, Dietzenbach, Germany). 14 Lysates were loaded on 10% sodium dodecyl sulphate polyacrylamide gels, and electrophoresis was performed and transferred to nitrocellulose membranes. Membranes were incubated overnight with antibodies directed to human STAT1, pSTAT1, STAT3, pSTAT3, STAT6 and pSTAT6 (Cell Signaling Technology, UK) at 4 °C. Anti-human ASH antibody (Elisabeth Kremmer, Munich, Germany) was used as a loading control. Secondary peroxidase-conjugated goat anti-rabbit IgG or goat antimouse IgG (Jackson ImmunoResearch, Baltimore, MD, USA) was incubated for 1 h at room temperature. Signal was detected using Amersham ECL prime Western blotting detection reagent (GE Healthcare Life Sciences, Freiburg, Germany) and quantified using Fujifilm luminescent Image Analyzer LAS-3000 (Fuji Photo Film, D€ usseldorf, Germany).
4
Western Blot Protein Analysis Protocol
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Cellular proteins were prepared with lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 [NP-40] [28324, Thermo Fisher Scientific], 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3PO4, and protease inhibitor [11 697 498 001, Hoffmann-La Roche, Basel, Switzerland]). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk (232100, BD Difco, BD Biosciences, Franklin Lakes, NJ, USA) in Tris-buffered saline with Tween 20 (TBST) (20605, Thermo Scientific Fisher), the membranes were incubated with primary antibodies overnight at 4°C on a rocker. After three washes in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (7076, 7074, Cell Signaling Technology) for 1 h at room temperature. The peroxidase activity was visualized by enhanced chemiluminescence using western blotting luminol reagent (sc-2048, Santa Cruz Biotechnology) and a Fujifilm luminescent image analyzer LAS-3000 (Fujifilm Life Sciences, Richmond, VA, USA). Quantification of western blot analysis was performed after retrieving the density of the bands using Scion Image software (Scion, Frederick, MD, USA) after more than three independent sets of experiments.
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