With density gradient centrifugation, cytosolic and nuclear fractions were separated after H9c2 cells from each group were lysed in RIPA buffer (Beyotime, Shanghai, China). Subsequently, the mixture was centrifuged for 15 min (12,000 r/min, 4°C) and the supernatant was collected, which was considered as the protein sample, before protein concentration was measured using Bicinchoninic Acid (BCA) Protein Assay Kit (Boster, Wuhan, China). After the protein samples were denatured, 20 μg of protein sample in each group was dissolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then the proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime, Shanghai, China). Following that, the PVDF membrane was blocked with 5% skim milk for 2 h and then incubated with anti-histone H3 (Abcam, ab215728, Cambridge, UK, 1:1000), anti-GAPDH (Abcam, ab8245, Cambridge, UK, 1:1000), anti-STAT3 (Proteintech, 10253-2-AP, Wuhan, China, 1:1000) and anti-p-STAT3 (Abcam, ab76315, Cambridge, UK, 1:500) primary antibodies at 4°C overnight. Next, the membrane was incubated with the secondary antibody (Proteintech, 10253-2-AP, Wuhan, China) at room temperature for 2 h. Finally, the protein bands were detected with the enhanced chemiluminescence method (Beyotime, Shanghai, China).
Ab215728
Manufactured by Abcam
Sourced in United Kingdom
Ab215728 is a laboratory equipment product offered by Abcam. It serves a core function, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Enhanced chemiluminescence method, by Beyotime (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Ab179461, by Abcam (1 mentions)
Ab5076, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab108424, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Rabbit anti sars cov 2 spike, by Sino Biological (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
4 protocols using ab215728
1
Cytosolic and Nuclear Protein Isolation
2
Protein Purification and Western Blotting
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Total protein was purified from the brain tissues of 14 days after MCAO in the absence or presence of 10 mg/kg Paeoniflorin treatment using lysis buffer and then centrifuged at 12,000 rpm for about 15 min. The protein concentration was measured with the BCA kit (Pierce, IL). The western blotting was carried out as the method reported by Zhu et al. (2019) (link). The cell was cultured with primary antibodies, anti-Iba-1 antibody (1 µg/ml; ab5076; Abcam, MA), anti-JNK antibody (1:1000; ab179461; Abcam, MA), anti-phosphorylated-JNK antibody (1:1,000; #9251; Cell Signaling Technology), anti-P65 antibody (0.5 µg/ml; ab16502; Abcam), anti-Histone H3 antibody (1:1,000; ab215728; Abcam) and anti-actin antibody (1:1000, ab6276, Abcam), at 4 °C overnight. After washing, horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated with membranes for 1 h.
3
Protein Expression Analysis by Western Blot
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Protein samples were collected with the RIPA buffer (Beyotime, China). Commercial kit (Invitrogen, USA) was used for the separation of the proteins in nucleus and cytoplasm. Then, BCA (Beyotime, China) kit was used for the determination of the concentration of proteins. Ten percent SDS PAGE gel was used for the separation of proteins. Then, proteins were transferred to the PVDF membrane (Millipore, USA) and the membranes were blocked with the BSA (Beyotime, China) followed by incubation with the primary antibodies at 4°C overnight. The primary antibodies used in this research were KLF1 (ABCAm, ab175372), E-cadherin (ABCAm, ab40772), Vimentin (ABCAm, ab92547), α-SMA (ABCAm, ab108424), Snail (ABCAm, ab216347), ZBTB7A (ABCAm, ab175918), c-myc (ABCAm, ab32072), β-catenin (ABCAm, ab32572), β-tubulin (ABCAm, ab18207), Histone H3 (ABCAm, ab215728) and β-actin (ABCAm, ab8226) antibodies. These antibodies were diluted with the BSA at the ratio of 1:1000. Then, membranes were incubated with the secondary antibodies for 2 hours. Finally, immunoreactive signals were measured with the western blotting substrate (Millipore, USA). The results of this experiment were analyzed with the Image J.
4
Immunofluorescence Staining of Lung Tissues and Neutrophils
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Lung tissues were harvested and used for IF staining [48 (link)]. Briefly, after incubation with blocking buffer (5% normal goat serum) for 10 min at room temperature, the slides were incubated with primary antibodies overnight at 4 °C. After five washes, the sections were incubated with secondary antibody at 37 °C for 1 h. In the in vitro experiment, neutrophils were allowed to attach to the coverslips and incubated for 4 h at 37 °C for immunostaining. The primary antibodies used for IF analysis included rabbit anti-MPO (Abcam, ab9535), rabbit anti-SARS-CoV-2 Spike (Sino Biological, 40589-T62), and recombinant anti-histone H3 (methyl K37) antibodies (Abcam, ab215728). Secondary antibodies included goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam) and goat anti-rat IgG H&L preadsorbed (Alexa Fluor® 647) (Abcam, ab150167).
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