Total protein was extracted from BMDMs by incubating the cells for 10 min at 4 ℃ in RIPA lysis buffer (Fdbio Science, Guangzhou, China) with additional protease inhibitor. After BCA protein determination (Fdbio Science), 5 × loading buffer (Fdbio Science) was added in protein lysates. Equal quantities of lysates were isolated by SDS-PAGE and transferred onto 0.22 um PVDF microporous membranes (Beyotime Institute of Biotechnology, Jiangsu, China). The membranes were blocked with 5% whole milk and then incubated with primary antibodies for 16 h at 4 °C. Afterwards, the membranes were incubated at room temperature for 60 min with the secondary antibodies. Target protein bands were visualized by FDbio-Dura ECL (Fdbio Science). Antibodies applied for western blot in this study were: rabbit anti-IGF2BP3 (Proteintech, Rosemont, USA, 1:1,000, 14642-1-AP), rabbit anti-iNOS (Proteintech, 1:1,000, 22226-1-AP), rabbit anti-CD206 (Proteintech, 1:1,000, 18704-1-AP), rabbit anti-IL-1β (Proteintech, 1:1,000, 16806-1-AP), mouse anti-IL-4 (Proteintech, 1:1,000, 66142-1-Ig), species-matched HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
Dura ecl
Manufactured by Fdbio
Sourced in China
The FDbio-Dura ECL is a laboratory equipment designed for enhanced chemiluminescence detection. It provides a durable and reliable solution for researchers in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Loading buffer, by Fdbio (1 mentions)
Rabbit anti igf2bp3, by Proteintech (1 mentions)
Rabbit anti inos, by Proteintech (1 mentions)
Rabbit anti cd206, by Proteintech (1 mentions)
Rabbit anti il 1β, by Proteintech (1 mentions)
Mouse anti il 4, by Proteintech (1 mentions)
Ripa lysis buffer, by Fdbio (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bca protein assay kit, by Fdbio (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
A19544, by Abcam (1 mentions)
Histone h3, by ABclonal (1 mentions)
Bca assay kit, by Fdbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
P16ink4a, by Abcam (1 mentions)
4 protocols using dura ecl
1
Western Blot Analysis of Macrophage Markers
2
Western Blot Analysis of Hugl-2 Expression
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Hugl‐2 protein expression was detected by western blotting, as described previously.40 After treatment with DAC, lysates were further centrifuged at 14 500 g for 15 minutes at 4°C, and the supernatants were collected and stored at −80°C. Total protein concentrations were determined using a BCA protein assay kit (Fdbio). Equal amounts of protein (100 μg) with loading buffer were separated on SDS‐polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes (Roche). The following antibodies were used: anti‐GAPDH (Proteintech) and anti‐Hugl‐2 (Abnova). Chemiluminescent signals were detected using Fdbio‐Dura ECL (Fdbio).
3
Western Blot Analysis of Protein Expression
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Cells cultured in 6-well dishes were, after lysed with 200μL of radioimmunoprecipitation assay (RIPA) buffer, and then added protease inhibitor and phosphatase inhibitor 2μL. Heating at 100 degrees for 15 min after additionally add 40μL 5 × Loding buffer(Vazyme Biotech, Nanjing, China). And then the protein samples additionally addgel electrophoresis (SDS-PAGE), and then were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated for 1 h at room temperature in shaking bed with 5% skim milk solution. And then membranes incubated for 16 h at 4℃ in shaking bed with primary antibodies. And then washed the membranes three times with TBST solution, 5 min per time. Finally the membranes were incubated for 1 h at room temperature in shaking bed with 5% skim milk solution and secondary antibodies diluted in it. Protein bands were exposed by FDbio-Dura ECL(FDbio science, Hangzhou, China). Antibodies used for western blotting were: Ferritin Heavy Chain Rabbit mAb( ABclonal, 1:1000, A19544), rabbit anti-P21 (Abcam, 1:2000, ab109520), SOX9 Rabbit mAb (ABclonal, 1:1000, A19710), ERK1/2 Polyclonal antibody (Proteintech 1:1000, 11257–1-AP), Phospho-ERK1/2 Polyclonal antibody (Proteintech 1:1000, 28733–1-AP).
4
Protein Extraction and Western Blot Analysis
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The protein in cells or ectosomes was extracted by RIPA lysate (Fdbio Science, Guangzhou, China) and ultrasonic lysis of cells and ectosomes, and protein degradation was inhibited by protein phosphatase inhibitor. The total protein levels in these samples were measured by BCA assay kit (Fdbio Science). After separation of samples by SDS-PAGE, they were transferred to a 0.22um PVDF membrane in the wet transfer method. At 4 °C on a shaker, blocking was performed on each membrane using 5% whole milk. After blocking, the membranes were further processed for immunoblotting using specific primary antibodies. Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).
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