Cell lysates were prepared in lysis buffer (150 mM NaCl, 50 mM Tris-Cl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 0.02% sodium azide, 1 mM sodium vanadate, protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride)). Protein concentrations were determined using a Bio-Rad protein assay. Ten to 50 μg protein was loaded in 4–12% Mini-PrROTEAN TGX Precast protein Gels (Bio-rad), and transferred to PVDF membrane. Membranes were blocked in 3% BSA for 1 h, incubated with the primary antibody overnight at 4 °C, washed, and incubated with the appropriate horseradish-conjugated secondary antibody for 1 h at room temperature. An enhanced chemiluminescent (ECL) kit was used to visualize the signal (Thermo Fisher). The Abs used for western blot analysis are listed in Supplementary Table 3 . Uncropped blots for western blot analysis are shown in Supplementary Fig. 11 .
Enhanced chemiluminescent ecl kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Enhanced chemiluminescent (ECL) kit is a laboratory equipment product designed to detect and quantify proteins in Western blot analyses. The kit uses a chemiluminescent substrate to generate a luminescent signal that is proportional to the amount of target protein present in the sample. The core function of the ECL kit is to provide a sensitive and reliable method for protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Cdna synthesis kit, by PCR Biosystems (1 mentions)
Sphingosine 1 phosphate (s1p), by Merck Group (1 mentions)
Magextractor rna kit, by Toyobo (1 mentions)
Revertra ace qpcr rt master mix with gdna remover kit, by Toyobo (1 mentions)
Tgf β, by Merck Group (1 mentions)
Ab109290, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab20272, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
4 protocols using enhanced chemiluminescent ecl kit
1
Western Blot Analysis of Protein Lysates
2
Quantitative RT-PCR Analysis of QKI-5 Expression
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To develop the RT-PCR blot, we first performed reverse transcription on total RNA samples using the cDNA synthesis kit (PB30.11 PCR Biosystems Ltd., Plymouth, PA, USA). The cDNA samples were then amplified using specific primers for the gene of interest and the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Primers of QKI-5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences are described in the previous study [24 (link)]. The amplified cDNA was resolved on a 1.5% agarose gel and visualized using the GelDoc XR + system (Bio-Rad, Hercules, CA, USA). The blot was developed using the Enhanced Chemiluminescent (ECL) kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The resulting image was captured using the ChemiDoc MP System (Bio-Rad, Hercules, CA, USA).
3
Modulating S1P/TGF-β Signaling Axis
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All treatments were administered at a concentration of 10 mM for 48 h of treatment at 37°C. S1P, W146, SB-431542 (SB4), SIS3 and TGF-β were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The primary anti-S1PR1, anti-TGF-β and anti-Smad3 antibodies (Cat. no. ab72806, ab31013, ab40854, respectively) were acquired from Abcam (Cambridge, UK). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were from Hyclone (GE Healthcare, Little Chalfont, UK). The secondary antibodies conjugated to horseradish peroxidase (Cat. no. ab6789) were purchased from Zhongshan Goldenbridge Bio (Beijing, China). The enhanced chemiluminescent (ECL) kit was obtained from Thermo Fisher Scientific (Shanghai, China). For Reverse-transcription quantitative polymerase chain reaction (RT-qPCR), the MagExtractor-RNA kit and ReverTra Ace qPCR RT Master Mix with gDNA Remover kit were all from Toyobo (Cat. no. FSQ-301, Tokyo, Japan). The lactate dehydrogenase (LDH) detection activity assay kit and the commercialized caspase-3 assay kit were purchased from Sigma-Aldrich (cat. no. CASP3F-1KT, Merck KGaA) and Biovision, Inc. (cat. no. 1533-100, Milpitas, CA, USA), respectively.
4
Western Blot Analysis of SOX1 Protein
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RIPA (Beyotime, Shanghai, China) was applied to isolate total protein. BCA (Thermo Fisher Scientific) was used to quantify the total protein. SDS-PAGE (10%) was used to separate proteins (40 μg per lane), and then proteins were transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with primary antibodies against SOX1 (1:1000, #ab109290, ABCAm, Cambridge, UK) and β-actin (1:5000, #ab8226, ABCAm, Cambridge, UK) at 4 °C overnight after being blocked with 5% skimmed milk for 1 h. After that, the membranes were incubated with secondary antibodies HRP-conjugated (1:5000, #ab20272, ABCAm, Cambridge, UK) for 1 h at room temperature. Protein bands were visualized using the enhanced chemiluminescent (ECL) kit (Thermo Fisher Scientific). β-actin was used for normalization. Image-Pro Plus 6.0 was applied for densitometric analysis.
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