Total tissue protein was extracted from cell lysates (Beyotime, Shanghai, China), and the protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit (Beyotime). Protein samples were electrophoresed on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk powder at room temperature for 2 h, incubated with appropriately diluted primary antibody at 4 °C overnight, rinsed 3 times, and incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 2 h. The bands were visualized with a fluorescence chemiluminescence imaging system (Clinx Science Instruments, Shanghai, China). Anti-TGF-β1, anti-α-SMA, anti-Fn, anti-GAPDH, anti-JAK2, anti-p-JAK2, anti-STAT3, and anti-p-STAT3 monoclonal antibodies were purchased from Affinity (Jiangsu, China). Anti-TGF-β-R2 antibodies were purchased from Proteintech. Antibody dilutions were purchased from Beyotime. HRP-labeled goat anti-rabbit and goat anti-mouse IgG antibodies were purchased from EarthOx (USA).
Fluorescence chemiluminescence imaging system
Manufactured by Clinx
Sourced in China
The Fluorescence chemiluminescence imaging system is a laboratory instrument designed to detect and analyze fluorescent and chemiluminescent signals. It is used for imaging and quantifying a variety of biological samples, such as proteins, nucleic acids, and cells.
Automatically generated - may contain errors
Lab products found in correlation
Cell lysate, by Beyotime (1 mentions)
Anti gapdh, by Affinity Biosciences (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Anti stat3, by Affinity Biosciences (1 mentions)
Anti tgf β1, by Affinity Biosciences (1 mentions)
Anti pjak2, by Affinity Biosciences (1 mentions)
Anti jak2, by Affinity Biosciences (1 mentions)
Anti α sma, by Affinity Biosciences (1 mentions)
Goat anti mouse igg peroxidase, by Merck Group (1 mentions)
Hybond c super, by GE Healthcare (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Goat anti mouse igg ha1006, by Huabio (1 mentions)
Ecl western blotting substrate, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
4 protocols using fluorescence chemiluminescence imaging system
1
Protein Quantification and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
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Equivalent amounts of each sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Super; GE Healthcare). The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween (PBST) and incubated for 2 h at room temperature with the primary antibodies at an appropriate dilution (anti-Flag, -Myc, -MDA5, and -RIG-I monoclonal antibodies [MAbs] at 1:1,000 and anti-hHB MAb at 1:500) (catalog no. F1804 and M4439 [Sigma-Aldrich] and catalog no. sc-134513, sc-48932, and sc-22718 [Santa Cruz]). The membranes were washed with PBST and then incubated with IRDye 800CW goat anti-mouse IgG(H+L), donkey anti-goat IgG(H+L), and goat anti-rabbit IgG(H+L) (catalog no. 926-32210, 926-32214, and 926-32211; LiCor BioSciences) or goat anti-mouse IgG-peroxidase (catalog no. A5278; Sigma) at 1:10,000 for 1 h at 37°C, and the blots were scanned using an Odyssey infrared imaging system (LiCor BioSciences) or a fluorescence/chemiluminescence imaging system (Clinx Science instruments).
3
Quantitative Western Blot Analysis
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Nearly, 20-30 mg kidney tissue was homogenized in SDS-lysis buffer (#7722, CST) containing 42 mM DTT following the user manual. The same samples were loaded to the SDS-PAGE gels and run at 100 v for 1 h 40 min at RT in Tris-Glycin-SDS containing buffer. Blots were blocked with 5% non-fat dry milk powder in tris-buffer saline containing Tween-20 (TBST) for 30 min at RT. The blots were incubated in primary antibody overnight at 4 °C. After primary antibody (all the antibodies were shown in supplementary Data 4 ) incubation, blots were washed three times with TBST. Horseradish peroxidase-labeled goat anti-rabbit IgG (HA1001, 1:2000 dilution; HuaBio, Hangzhou, China) or goat anti-mouse IgG (HA1006, 1:2000 dilution; HuaBio, Hangzhou, China) were probed for 1 h at RT prepared in non-fat dry milk containing Tween-20. Finally, blots were washed with TBST for three minutes each 10 min at RT, and observed through Odyssey infrared imaging system. (Fluorescence Chemiluminescence Imaging System, Clinx Science, Shanghai, China) and quantified through utilizing ImageJ software (version 6.0; Wayne Rasband, National Institutes of Health, USA).
4
Western Blot Analysis of SNAP25 Expression
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Subsequently, 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed to separate the proteins, and a western blot analysis was conducted to determine the expression of the proteins of interest, based on the experimental protocols documented in a previous study. Primary antibodies against SNAP25 (1:1000, Wanleibio, Shenyang, China) and β-actin (1:1000, ZSGB-BIO, Beijing, China) were used to probe corresponding proteins. Specific protein bands were detected with an ECL western blotting substrate (Beyotime, Shanghai, China) using the Fluorescence/Chemiluminescence Imaging System (CLINX Science instruments, Shanghai, China). and analyzed using ImageJ software. Each experiment was repeated at least three times.
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