Cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Beyotime, Haimen, China). Total and nuclear proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). Protein samples (50 μg) were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBST containing 5% fat-free milk at room temperature for 1 h. After washing with TBST, the membranes were probed with primary antibodies against C/EBPα, FABP4, PPARγ, RhoA, ROCK1, ROCK2, ERK1/2, β-actin, PCNA (all Proteintech, Wuhan, China), and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed in TBST and incubated with secondary antibody (Proteintech, Wuhan, China). The signals were developed by ECL reagents (Beyotime, Haimen, China).
Ecl reagent
Manufactured by Beyotime
Sourced in China, United States
ECL reagent is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It produces a luminescent signal when exposed to horseradish peroxidase (HRP), which is commonly used to label antibodies in immunodetection procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (71 mentions)
Ripa buffer, by Beyotime (41 mentions)
Ripa lysis buffer, by Beyotime (36 mentions)
Bca protein assay kit, by Beyotime (33 mentions)
Bca kit, by Beyotime (27 mentions)
Ab205718, by Abcam (15 mentions)
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Ab8245, by Abcam (7 mentions)
Ab32503, by Abcam (7 mentions)
Ab9485, by Abcam (7 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (6 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Chemidoc xrs system, by Bio-Rad (6 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (6 mentions)
Gel doc xr imaging system, by Bio-Rad (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Protease inhibitor, by Beyotime (5 mentions)
Image lab software, by Bio-Rad (5 mentions)
Ripa buffer, by Solarbio (5 mentions)
263 protocols using ecl reagent
1
Western Blot Analysis of Adipogenic Markers
2
Protein Expression Analysis in Lipid-Treated Cells
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After treatment with stearate, oleate or linoleate, cells were collected and total proteins were lysed using RIPA lysis buffer (Beyotime, Nanjing, China). The homogenates were combined with equal volumes of SDS sample buffer, and the proteins were separated by electrophoresis on a 5~12% polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20, followed by overnight probing with the following primary antibodies: (1) CD36 (N-15) antibody (sc-5522, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (2) ACACA (T-18) antibody (sc-26817, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3) DGAT1 antibody (ab59034, 1:500, Abcam, Cambridge, MA, USA), (4) SREBP1 (C-20) (sc-366, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (5) PPARγ (T-18) antibody (ab19481, 1:500, Abcam, Cambridge, MA, USA), (6) β-actin (C4) antibody (sc-47778, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin was intended to serve as a loading (internal) control. After washing, membranes were incubated with secondary antibody (ABR, Golden, CO, USA) and conjugated to HRP. The chemiluminescent signal was detected using ECL reagents (Beyotime, Nanjing, China) and bands were quantified by image processing software (Image Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA).
3
Western Blot Analysis of Lipogenic Proteins
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Protein was extracted using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein (20 µg) was separated by 8% SDS-PAGE and transferred on to PVDF membrane. The membranes were incubated with antibodies of ChREBP (1:1,000, Abcam, cat. no. ab92809), fatty acid synthase (FASN; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. PB0909), acetyl-CoA carboxylase (ACC; 1:500, Wuhan Boster Biological Technology, Ltd., cat. no. BM4414), AMPK (1:1,000, Cell Signaling Technology, Inc., cat. no. 2532), phosphorylated (p)-AMPK (Thr172; 1:1,000, Cell Signaling Technology, Inc., cat. no. 2535), AKT (1:1,000, Cell Signaling Technology, Inc., cat. no. 9272), p-AKT (Ser473; 1:1,000, Cell Signaling Technology, Inc., cat. no. 9271), GAPDH (1:2,000, Wuhan Boster Biological Technology, Ltd., cat. no. BM1623) and FOXO1 (1:500, Wuhan Boster Biological Technology, Ltd., BM4249) overnight at 4°C. Then, membranes were incubated with rabbit anti-mouse lgG (cat. no. BA1048) and goat anti-rabbit lgG antibodies (cat. no. BA1039; both from Wuhan Boster Biological Technology, Ltd.). Finally, the results were detected by ECL reagents (Beyotime Institute of Biotechnology) and semi-quantified by densitometry using the Tanon 5200 automatic chemiluminescent imaging system (Tanon Science and Technology Co., Ltd).
4
Western Blot Analysis of PM2.5-induced Apoptosis
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PM2.5-treated cells were harvested, lysed in RIPA buffer (Beyotime, Shanghai, China) on ice for 30 min, and centrifuged at 12,000 r/min (centrifugal radius: 9.6 cm) for 15 min at 4°C. The protein was quantified with a BCA protein assay kit (Dahui Bio, Guangzhou, China), separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech., England, UK) by semidry electrophoretic transfer at 300 mA for 30–90 min. The membranes were washed with Tris-buffered saline (TBS), blocked with 5% skim milk in TBS with Tween-20 (TBST) for 1 h at room temperature, and then incubated with primary antibodies (anti-β-actin, 1:400, anti-cytochrome-c, 1:5000, anti-caspase-3, 1:5000, anti-caspase-9, 1:2000, Abcam, Cambridge, UK) diluted in TBST for 2 h at room temperature. The probed membranes were washed three times with TBST for 10 min each, and then incubated with secondary antibodies for 2 h at room temperature. Immunoreactive bands were detected with ECL reagents (Beyotime, Shanghai, China) and exposed to X-ray film. β-actin served as a loading control for total protein and showed no differences between groups. Densitometric quantification was performed in Image J (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Inflammatory Markers
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BV2 cells and spinal cord segments (1 cm) were washed with PBS and lysed with RIPA buffer at 4°C for 30 min. Lysates were centrifuged at 12,000
g for 20 min at 4°C to remove the insoluble pellets. Protein concentration was determined using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Temecula, USA). After incubation with 5% skim milk for 1 h, the membranes were incubated with primary antibodies including anti-iNOS (1:1000, ab283655; Abcam, Cambridge, UK), anti-Arg-1 (1:1000, ab233548; Abcam), anti-MIF (1:1000, PA5-27343; Invitrogen), anti-ERK1/2 (1:10000, ab184699; Abcam), anti-p-ERK1/2 (1:2000, 4370T; Cell Signaling Technology, Danvers, USA), anti-CD74 (1:1000, ab289885; Abcam), and anti-GAPDH (1:2000, ab8245; Abcam). The next day, the membranes were incubated with the corresponding HRP-conjugated goat anti-rabbit IgG (1:3000, ab205718; Abcam) or goat anti-mouse IgG (1:3000, ab6789; Abcam) secondary antibodies for 2 h at room temperature. The bands were then detected using ECL reagents (Beyotime, Beijing, China).
g for 20 min at 4°C to remove the insoluble pellets. Protein concentration was determined using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Temecula, USA). After incubation with 5% skim milk for 1 h, the membranes were incubated with primary antibodies including anti-iNOS (1:1000, ab283655; Abcam, Cambridge, UK), anti-Arg-1 (1:1000, ab233548; Abcam), anti-MIF (1:1000, PA5-27343; Invitrogen), anti-ERK1/2 (1:10000, ab184699; Abcam), anti-p-ERK1/2 (1:2000, 4370T; Cell Signaling Technology, Danvers, USA), anti-CD74 (1:1000, ab289885; Abcam), and anti-GAPDH (1:2000, ab8245; Abcam). The next day, the membranes were incubated with the corresponding HRP-conjugated goat anti-rabbit IgG (1:3000, ab205718; Abcam) or goat anti-mouse IgG (1:3000, ab6789; Abcam) secondary antibodies for 2 h at room temperature. The bands were then detected using ECL reagents (Beyotime, Beijing, China).
6
Western Blot and IP Analysis of HSP70 and HSP90
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The jejunum tissue was lysed with western blot and immunoprecipitation (IP) lysis buffers (P0013, Biosharp, China) containing protease inhibitors (Beyotime, China). According to the instructions of the kit, the protein concentration of all samples was determined by the BCA protein determination kit (Beyotime, China). Initially, 20 μg of lysate was electrophoresed through 10% or 12% SDS-PAGE gels (Solarbio, China). Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Cytiva, Marlborough, MA, USA). At room temperature, the membrane was blocked with 5% BSA for 1 h and incubated with the first antibody at 4°C overnight and with the second antibody against rabbit IgG (1:2000, Santa Cruz, USA) for 1 h at room temperature. The HSP 70 (HSP70, 1:800) and HSP 90 (HSP 90, 1:1000) were detected, and β-actin (1:5000) was used as the analysis reference. BOX Chemi XX9 imager (Syngene, Cambridge, UK) was used to detect protein signals with potentiated chemiluminescence (ECL) reagents (Beyotime, China). ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the relative density of bands.
7
Western Blot Analysis of Lipid Metabolism Proteins
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Following the incubation with the different treatments, SDS buffer was used to extract total proteins from the harvested cells which were washed twice and collected in ice-cold PBS. The untreated cells were used as control. Equal amounts of total proteins (100 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE ) (6%) and transferred to a Polyvinylidene Fluoride (PVDF ) membrane. After blocking with a mixture of 5% skimmed milk/Tris-buffered saline Tween 20 (TBST ), the membranes were incubated overnight at 4˚C with the primary antibody rabbit against sterol regulatory element-binding proteins-1 (SREBP1 ) (bs-1402R), carnitine palmitoyltransferase (CPT1A ) (bs-2047R), microsomal triglyceride transfer protein (MTP) (bs-5083R) antibodies (1:1,000; Beijing Biosynthesis Biotechnology, China); antibody information was listed in Supplement materials S-Table2 . Following three consecutive washes in TBST (0.05%), the membranes were incubated with the goat anti-rabbit horseradish peroxidase-conjugated IgG at 1:2,000 (Beijing Biosynthesis Biotechnology) for another 2 h at room temperature. The results were normalized to α-Tubulin (bs-0519R) (Beijing Biosynthesis Biotechnology) protein levels. Protein expression levels were finally visualized using enhanced chemiluminescence (ECL ) reagents (Beyotime Institute of Biotechnology, China).
8
Protein Detection by Western Blot
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Proteins were extracted by RIPA buffer (Beyotime). Samples were separated by SDS‐PAGE and then transferred to PVDF membranes (EMD Millipore). The membranes were blocked with skimmed milk powder, followed by incubated with primary antibodies and secondary antibodies. The bands were formed with ECL reagents (Beyotime) and analysed with ImageJ 6.0 software.
9
ADAM12 Glycoprotein Enrichment and Western Blot
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Cells were treated with lysis buffer (Beyotime Institute of Biotechnology, China) for 45 min on ice. ADAM12 enrichment from HCC cell lysates was performed for glycoproteins by binding to concanavalin A agarose (Sigma Aldrich; 25 mL resin/500 mL lysate). Equal amounts of protein were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA) at 90–100 mA for 1.5–2 h. Membranes were blocked with PBS-0.05% Tween 20 containing 5% nonfat dry milk for 1 h and incubated with primary antibody overnight at 4°C. Membranes were then washed three times with PBS-0.05% Tween 20 and incubated with secondary antibody for 2 h at room temperature. Blots were developed using an ECL reagents (Beyotime Institute of Biotechnology, China). Each experiment was repeated at least three times. Antibodies used in the experiments included rabbit anti-human TSPAN8 antibody (Abcam, USA), rabbit anti-human MMP-2 antibody (BioWorld, USA), and rabbit anti-human MMP-9 antibody (BioWorld, USA) followed by a 1:1000 dilution of HRP-conjugated goat anti-rabbit IgG F(ab')2 antibody (Beyotime Institute of Biotechnology, China), as well as rabbit anti-human ADAM12m antibody (Sigma-Aldrich, USA) followed by a 1:1000 dilution of HRP-conjugated goat anti-rabbit IgG F(ab')2 antibody (Beyotime Institute of Biotechnology, China).
10
Protein Expression Analysis of Mammary Tissue
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Mammary tissue samples were homogenized in RIPA lysis buffer (Beyotime, Nanjing, China). Extracted protein samples were heated in water at 100 °C for 5 min and separated by electrophoresis (Bio-Rad, Richmond, CA) in 12% SDS-PAGE gel before being electroblotted (Bio-Rad) onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). After polyvinylidene fluoride membranes were blotted with Tris buffer containing 5% fat-free dry milk and 0.05% Tween-20 (TBST; 0.05% Tween 20, 100 mmol/L of Tris-HCl, and 150 mmol/L of NaCl, pH 7.5) for 1 h at 25 °C, they were rinsed in TBST 4 times and incubated overnight at 4 °C with primary antibodies: EAAT3 (Sigma Aldriche, sc-25658), ASCT1(Sigma Aldriche, sc-50801), and SNAT2 (Sigma Aldriche, sc-166366) with ratios of 1:1,000, 1:800, and 1:1,000 respectively. Rabbit IgG (5 μg/mL; Sigma) was used as the primary antibody for controls. After washing, membranes were incubated with secondary antibody (Amersham Biosciences). The chemiluminescent signal was detected by using ECL reagents (Beyotime, Nanjing, China), and bands were quantified by Image Processing Software (Image Pro Plus6.0).
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