Cells and ventricular tissue were lysed with RIPA buffer (Sigma–Aldrich, USA), and the protein concentrations of the lysates were determined using BCA Protein Assay Reagent (Pierce Biotechnology, USA). Total protein samples (40 μg) were separated electrophoretically, transferred to a PVDF membrane and probed using monoclonal primary antibodies against PI3K (1:5000, Abcam, ab139307), p-PI3K (1:1000, Abcam, ab32089), AKT (1:1000, Abcam, ab179463), p-AKT (1:1000, Cell signaling, 4060s), KDM5A (1:5000, Abcam, ab194286), IGF1 (1:1000, Abcam, ab223567), MYH11 (1:1000, Abcam, ab82541), TGFB3 (1:1000, Abcam, ab15537), and β-actin (1:500, Abcam, ab5694), followed by an HRP-conjugated secondary antibody (1:5000; Abbiotec, USA). Bands were exposed using an ECL kit (Bio–Rad, USA) and analyzed using Image-Pro Plus software. LY294002 (20 μmol/L, Cell signaling, 9901s) is an inhibitor of PI3K, and ARQ-092 (10 μmol/L, Abcam, ab235550) is an inhibitor of AKT.
Ab223567
Manufactured by Abcam
Sourced in United States
Ab223567 is a primary antibody for use in immunoassays. It is a purified polyclonal antibody raised in rabbit against a specific target antigen. The antibody is supplied in a buffered solution.
Automatically generated - may contain errors
Lab products found in correlation
Ab15537, by Abcam (2 mentions)
Ab82541, by Abcam (2 mentions)
Ab194286, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab32089, by Abcam (1 mentions)
Ab179463, by Cell Signaling Technology (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Abbiotec (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
Ab26414, by Abcam (1 mentions)
Ab189841, by Abcam (1 mentions)
Ab188570, by Abcam (1 mentions)
Ab22555, by Abcam (1 mentions)
4 protocols using ab223567
1
Protein Expression Analysis in Cardiac Tissue
2
Immunofluorescence Staining of Myocardial Tissue
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CFs were fixed with PBS/paraformaldehyde (4%) for 10 min and permeabilized with PBS/Triton X-100 (0.5%) for 5 min. Then, they were incubated with anti-vimentin antibody (1:50, Abcam, ab92547) at 4 °C overnight. Cy3-conjugated secondary antibodies (Invitrogen, A10522) were used at 1:100. Nuclei were counterstained with DAPI (Sigma–Aldrich, USA).
The procedure for immunofluorescence staining of myocardial tissue sections was as described in our previous study (17 (link)). The sections were incubated with vimentin (1:500, Abcam, ab92547), KDM5A (1:2000, Abcam, ab194286), IGF1 (1:400, Abcam, ab223567), MYH11 (1:1000, Abcam, ab82541), and TGFB3 (1:1000, Abcam, ab15537) overnight at 4 °C. Then, the sections were incubated with Alexa Fluor 488 donkey anti-mouse immunoglobulin G (Abcam, ab150105), Alexa Fluor 594 donkey anti-rabbit immunoglobulin G (Abcam, ab150156) or Alexa Fluor 633 donkey anti-goat immunoglobulin G (Invitrogen, A-21082). The nuclei were counterstained with DAPI (2.5 μg/ml in PBS; Molecular Probes).
The procedure for immunofluorescence staining of myocardial tissue sections was as described in our previous study (17 (link)). The sections were incubated with vimentin (1:500, Abcam, ab92547), KDM5A (1:2000, Abcam, ab194286), IGF1 (1:400, Abcam, ab223567), MYH11 (1:1000, Abcam, ab82541), and TGFB3 (1:1000, Abcam, ab15537) overnight at 4 °C. Then, the sections were incubated with Alexa Fluor 488 donkey anti-mouse immunoglobulin G (Abcam, ab150105), Alexa Fluor 594 donkey anti-rabbit immunoglobulin G (Abcam, ab150156) or Alexa Fluor 633 donkey anti-goat immunoglobulin G (Invitrogen, A-21082). The nuclei were counterstained with DAPI (2.5 μg/ml in PBS; Molecular Probes).
3
Protein Expression Analysis in IVD Tissues
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The RIPA buffer (Solarbio Life Sciences, China) was used to extract the total protein of samples from IVD tissues, according to the protocol. The BCA Protein Assay Kit (Thermo Fisher Scientific) was used to measure protein contant. Approximately 25 μg of total protein was boiled, separated using 12% SDS-PAGE, and blotted onto PVDF membranes. Then the membranes were sealed with 5% skimmed milk for 2 h, incubated with primary antibodies against IGF-1 (1:1000, ab223567, Abcam, Cambridge, MA, USA), TGF-β (1:500, ab92486, Abcam), SDF-1 (1:1000, ab18919, Abcam), CCL-5 (1:1000, ab189841, Abcam), SOX9 (1:1000, ab26414, Abcam), Aggrecan (1:1000, ab3778, Abcam), Collagen I (1:1000, ab233080, Abcam), Collagen II (1:1000, ab188570, Abcam) and GAPDH (1:5000, ab22555, Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were visualized using a chemiluminescence reagent (Millipore, USA). GAPDH was the internal reference. The relative expressions of proteins were normalized to GAPDH using Image-Pro software. The antibody information used in this study were as follows.Western blot experimental results of the original film in the supplementary file 1 .
4
Protein Expression Analysis in Cellular Stress Response
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Tissue samples or RSC96 cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitor cocktails. Total protein concentration was determined by BCA kit (TransGen, Beijing, China). Equal amounts of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking with nonfat dry milk for 2 h at room temperature, the membranes were incubated with primary antibodies (β-actin from mouse (100166-MM10), 1:1000, SinoBiological, Beijing, China; GRP78 from rabbit (3183S), 1:1000, CST, MA, USA; PERK from mouse (3192S), 1:800, CST; p-PERK from rabbit (3179S), 1:600, CST; ATF4 from rabbit (11815S), 1:800, CST; Bax from rabbit (2772T), 1:800, CST; IGF-1 from rabbit (ab223567), 1:1000, Abcam, MA, USA; and CHOP from mouse (2895S), 1:800, CST) overnight at 4 °C. The membranes were subsequently incubated with horseradish peroxidase (HRP)—labeled secondary antibodies (Goat anti-rabbit IgG (ab205719), Goat anti-mouse IgG (ab205718); 1:5000, Abcam) for 2 h at room temperature. Bands were visualized using an enhanced chemiluminescence Kit and an image system (Gel, BioRad, USA). β-actin served as a loading control to normalize the data. The relative expression level of p-PERK was shown as p-PERK/total PERK.
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