The protein expressions of PPARβ, α-SMA, Vimentin, COL1A, cyclin D1, bcl-2, and bax in HS fibroblasts with various treatments were detected with western blot assay based on the manufacturer's instructions. Firstly, HS fibroblasts were lysed by pre-cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors. Extracted proteins (40 μg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was sealed with 5% non-fat milk for 1 h at room temperature, followed by incubation with primary antibody against, PPARβ (1:500, sc-74517, Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-SMA (1:1000, ab7817, Abcam, Cambridge, UK), Vimentin (1:1000, ab92547, Abcam) and collagen type 1 α1 (COL1A; 1:1500, ab6308, Abcam), cyclin D1 (1:1000, ab134175, Abcam), bcl-2 (1:1000, ab32124, Abcam), bax (1:1000, ab69643, Abcam), and GAPDH (1:1000, ab8245, Abcam) at 4°C overnight. The membranes and horseradish peroxidase (HRP)-conjugated secondary antibody were incubated at 37°C for 2 h. The protein bands were detected with an ECL detection kit (Thermo Fisher Scientific).
Ab69643
Manufactured by Abcam
Sourced in United Kingdom
Ab69643 is a laboratory reagent used for scientific research. It is a primary antibody that can be used to detect and study a specific target protein or antigen. The core function of this product is to bind to and identify the target of interest within a sample. Further details on the intended use or specific applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ab32124, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab152126, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab7817, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab18265, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Ab32445, by Abcam (1 mentions)
Ab32522, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
Ab2001, by Abcam (1 mentions)
Ab2014, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
4 protocols using ab69643
1
Protein Expression Analysis in HS Fibroblasts
2
Podocyte Protein Expression Analysis
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Podocytes were lysed using pre-cold RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) with protease inhibitors (Sigma, St Louis, MO, USA). And then, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate these podocytes lysates, whereafter, these isolated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany). The membranes were sealed with 5% nonfat milk for 1 h at room temperature, and incubated with primary antibodies against E2F3 (ab152126, 1 : 2000 dilution, Abcam, Cambridge, UK), B-cell lymphoma-2 (Bcl-2, ab32124, 1 : 1000 dilution, Abcam), FN (ab18265, 1 : 1000 dilution, Abcam), Bcl-2 related X protein (Bax, ab69643, 1 : 1000 dilution, Abcam), α-SMA (Cat. 19245, 1 : 1000 dilution, Cell Signaling Technology, Danvers, MA, USA), Col I (Cat. 84336, 1 : 1000 dilution, Cell Signaling Technology) or β-actin (Cat. 3700, 1 : 2000 dilution, Cell Signaling Technology) overnight at 4 °C, following by incubation with horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1 : 10 000 dilution, Abcam) were incubated at 37 °C for 1 h. Protein bands were detected using an ECL reagent (Pierce Biotechnology, Rockford, IL, MA, USA).
3
Apoptosis Signaling Proteins in Podocytes
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Protein expression levels of E2F3, Bcl-2 related X protein (Bax), B-cell
lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1),
C-caspase3, C-caspase7, and C-caspase9 were measured using western blot assay in
podocytes under different treatments. Transfected podocytes (2×106cells/well) were lysed using pre-cold RIPA buffer (Beyotime Institute of
Biotechnology) according to the manufacturer instructions. Podocyte lysates were
separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then isolated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane (Millipore, USA). After blocking the proteins with 5%
non-fat milk in the membranes for 1 h at room temperature, membranes were
incubated with primary antibodies against E2F3 (ab152126, 1:2000 dilution), Bad
(ab32445, 1:1000 dilution), Bcl-2 (ab32124, 1:1000 dilution), Bax (ab69643,
1:1000 dilution), APAF1 (ab2001, 1:5000 dilution), C-caspase3 (ab4051, 1:1000
dilution), C-caspase7 (ab32522, 1:1000 dilution), C-caspase9 (ab2014, 1:1000
dilution), and GAPDH (1:1000, ab9485) (all from Abcam, UK) overnight at 4°C. The
horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1:10,000
dilution, Abcam) was then incubated with the membranes at room temperature for 1
h. Finally, an ECL detection kit (Pierce Biotechnolgy, USA) was used to detect
the protein bands.
lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1),
C-caspase3, C-caspase7, and C-caspase9 were measured using western blot assay in
podocytes under different treatments. Transfected podocytes (2×106cells/well) were lysed using pre-cold RIPA buffer (Beyotime Institute of
Biotechnology) according to the manufacturer instructions. Podocyte lysates were
separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then isolated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane (Millipore, USA). After blocking the proteins with 5%
non-fat milk in the membranes for 1 h at room temperature, membranes were
incubated with primary antibodies against E2F3 (ab152126, 1:2000 dilution), Bad
(ab32445, 1:1000 dilution), Bcl-2 (ab32124, 1:1000 dilution), Bax (ab69643,
1:1000 dilution), APAF1 (ab2001, 1:5000 dilution), C-caspase3 (ab4051, 1:1000
dilution), C-caspase7 (ab32522, 1:1000 dilution), C-caspase9 (ab2014, 1:1000
dilution), and GAPDH (1:1000, ab9485) (all from Abcam, UK) overnight at 4°C. The
horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1:10,000
dilution, Abcam) was then incubated with the membranes at room temperature for 1
h. Finally, an ECL detection kit (Pierce Biotechnolgy, USA) was used to detect
the protein bands.
4
Immunocytochemical Evaluation of Protein Expression
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To evaluate the protein expression, immunocytochemistry (ICC) technique was applied. After 5 days of coculture, the islets in all groups were fixed in 4% paraformaldehyde (Gibco, Germany) and embedded in low melt agar (Sigma, Germany). In the direct cocultured group, both islet and MSCs were fixed and separated from the plate with a scraper. After preparing 5 μm sections and deparaffinization, the slides were subjected to ICC using primary antibodies against Bcl-2 (Abcam, France, #ab115807), Bax (Abcam, France, #ab69643), active caspase-3 (Abcam, France, #ab32042), HIF-1α (Medaysis, USA, #RM0374), and p53 (Dako, USA, #M7001). HRP-secondary antibody (Abcam, France, #ab6717) was used for detection after overnight incubation with primary antibodies. Positive cells for protein expression appeared with 3,3′-diaminobenzidine (Sigma, Germany, #D12384) staining and counterstained by hematoxylin (Sigma, Germany, #H3136). The protein expression rate was calculated by the H-score method using the following formula: H − score = 1 × (%mild staining) + 2 × (%moderate staining) + 3 × (%strong staining) [3 (link), 28 (link)].
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