According to the manufacturer’s instructions, all proteins were collected using a RIPA buffer (Sigma, USA) and separated using SDS-PAGE (Bio-Rad, USA). The proteins were then transferred onto a PVDF membrane (Millipore, USA), and 5% fat-free milk was used to block the membrane. Incubation with the primary antibody happened overnight, followed by incubation with the respective secondary antibody (two hours). The results were visualised using an enhanced ECL system (Tanon, China). The antibodies used, with respective dilutions, were RAD23B (Abcam, ab194273, 1:1000), KLF2 (Abcam, ab203591, 1:1000), IgG (Abcam, ab150165, 1:500), and GAPDH (Abcam, ab8245, 1:5000).
Ab203591
Manufactured by Abcam
Sourced in United Kingdom
Ab203591 is a monoclonal antibody that recognizes an epitope present on the human SOD2 protein. The antibody can be used for various research applications, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab81289, by Abcam (2 mentions)
Ab150165, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Nb600 594, by Novus Biologicals (1 mentions)
Irdye 680lt, by LI COR (1 mentions)
A5228, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemiluminescence, by Bio-Rad (1 mentions)
Ab235585, by Abcam (1 mentions)
Confocal microscopy, by Leica camera (1 mentions)
Mca4750, by Bio-Rad (1 mentions)
Eclipse ni e, by Nikon (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab106629, by Abcam (1 mentions)
Blocking buffer, by Agilent Technologies (1 mentions)
16 protocols using ab203591
1
Protein Expression and Visualization
2
Western Blot Analysis of Fibrosis Markers
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Cell pellets were extracted in RIPA lysis buffer (P0013C, Beyotime, China) containing protease inhibitor cocktail (539131, Calbiochem, USA). Protein concentrations were determined by a BCA kit (23225, Pierce, USA). Equal amounts of cell extracts (30 μg total protein) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked in 5% non-fat dried milk in TBS-T at room temperature for 2 h and incubated with indicated primary antibodies at 4 °C overnight. The primary antibodies used in the study were: anti-α-SMA (1 µg/mL, A5228, Sigma-Aldrich), anti-collagen I (1 µg/mL, 600-401-103, Rockland), anti-collagen III (1 µg/mL, NB600-594, Novus), anti-Klf2 (2 μg/mL, ab203591, Abcam), anti-GAPDH (1 µg/mL, 5174, Cell signaling technology). The membranes were washed three times with TBS-T for 10 min, and then incubated with IRDye-680LT Goat Anti-Mouse IgG (H+L) (20 ng/mL,925-68020, Li-Cor, USA) or IRDye-800CW Goat Anti-Rabbit IgG (H+L) (20 ng/mL, 925-32211, Li-Cor, USA) at room temperature for 1 h. After washing in TBS-T for another three times, the membranes were detected by the Odyssey infrared imaging system (Li-Cor, USA). Experiments were repeated three times and the target protein level was quantified by Image J and normalized to internal control.
3
Quantifying Protein Expression Patterns
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After RIPA and PMSF lysis of tissues (50 mg) or cells, protein concentrations were measured using the BCA protein assay kit. Western blots were performed using standard protocols. Immunostaining was performed with antibodies against KLF2 (ab203591, 1:1000, Abcam), GJA1 (ab235585, 1:1000, Abcam), PCNA (2586, 1:2000, Cell Signaling Technology), and ACTB (60008, 1:2000, Proteintech). The intensities of protein bands were visualized using chemiluminescence (Bio-Rad, USA).
4
Immunohistochemical Analysis of Azoospermia Testes
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Obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) patients were used and classified according to WHO diagnostic criteria without karyotype abnormality and AZF microdeletion.3 (link) Testicular biopsies from OA or NOA patients who underwent microdissection and testicular sperm extraction were obtained from Hunan Guangxiu Hospital affiliated to Hunan Normal University. Immunohistochemistry was performed on 5 μm testis tissue sections using standard protocols. Immunostaining was conducted with antibodies against KLF2 (ab203591, 1:100, Abcam) and UCHL1 (MCA4750, 1:100, Bio-Rad). The positive cells were observed under a fluorescence microscopy or confocal microscopy (Leica, Germany). To evaluate OA and NOA patients’ testicular histology, sections were stained with haematoxylin and eosin (H&E).
5
Immunofluorescence detection of KLF2 protein
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Mesio-distal sections were incubated with rabbit anti-KLF2 polyclonal primary antibody (1:200 dilution; ab203591; Abcam, Cambridge, UK) overnight at 4 °C. The sections were then incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG; 1:200 dilution; ab150077; Abcam) and covered with Prolong Gold Reagent with DAPI (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). The sections were visualized using an Eclipse NiE automated upright fluorescence microscope (Nikon).
6
Immunofluorescence Staining of Cell Markers
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Cells grown on glass coverslips or tissue sections were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were washed twice with PBS. Blocking buffer (DakoCytomation, Glostrup, Denmark) was added for 30 min, and samples were then stained with primary antibodies and goat anti-mouse IgG/Alexa Fluor (Bioss Antibodies, bs-0296G-AF647, bs-0368G-AF488, 1:200 dilution). The following primary antibodies were used: αSMA (Proteintech, 55135-1-AP, 1:200 dilution), CD34 (Abcam, ab81289, 1:200 dilution), KLF2 (Abcam, ab203591, 1:100 dilution), KLF4 (Abcam, ab106629, 1:100 dilution), and Claudin5 (Abcam, ab15106, 1:100 dilution), ZO-1 (Cell Signaling, #8193, 1:1000 dilution), occludin (Abcam, ab216327, 1:100 dilution), CK (Abcam, ab191208, 1:200 dilution).
7
Chromatin Immunoprecipitation Assay for Transcription Factors
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Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT Express kit (Active Motif)33 (link), according to the manufacturer’s instructions. In brief, HUVEC were cross-linked for 10 min with 1% formaldehyde. Chromatin was sheared using a Bioruptor UCD-200 ultrasound sonicator (Diagenode) and immunoprecipitated with 3 μg antibody to ERG (sc-354X, Santa Cruz Biotechnology), KLF2 (rabbit, ab203591, Abcam), H3K27Ac (rabbit, 39133, Active Motif) or p300 (mouse, ab14984, Abcam). The respective negative controls were rabbit IgG (PP64, Chemicon, Millipore) and mouse IgG (12- 371, Millipore). Immunoprecipitated DNA was then used as template for quantitative PCR using primers specific for genomic loci and listed in Supplementary Table 5 .
8
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were carried out in terms of the manual of manufacturer (Cell Signaling Technology). Briefly, cells were cross-linked with 1% formaldehyde (Cell Signaling Technology), and formaldehyde was inactivated by the addition of 1M glycine. Chromatin extracts containing DNA fragments with an average size of 150–900 bp were immunoprecipitated using anti-KLF2 antibody (ab203591, 1:30, Abcam) or IgG (Cell Signaling Technology). The precipitated DNA was analyzed via real-time PCR.
9
Immunoblotting of Zebrafish Cardiac Proteins
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Primary antibodies, such as anti‐cleaved P53 (ab1101; Abcam, Shanghai, China), anti‐AMPK alpha 1 (ab32047; Abcam), anti‐KLF2 (ab203591; Abcam) and anti‐glucose transporter (GLUT)12 (ab75441; Abcam) were obtained. The anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; DH0261#) was purchased from Donghuan Biotech Co., Ltd (Shanghai, China). All antibodies were diluted according to the manufacturer’s instructions, and immunoreactivity was visualized using a chemiluminescence detection kit (DH0101). Dissection of zebrafish cardiac muscle was manually homogenized and then lysed in tissue lysis buffer with protease inhibitor (Roche, Basel, Switzerland).
10
ChIP Analysis of Transcription Factors in Dental Pulp Stem Cells
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ChIP analysis was performed by using Imprint® Chromatin Immunoprecipitation Kit (Sigma Aldrich Corporation, CHP1 96RXN) following manufacturers’ protocol. In brief, after chromatin cross-linking with 1% formaldehyde and DNA shearing, chromatin-protein complexes were immunoprecipitated from BGP + LAA-treated DPSCs, compared to undifferentiated DPSCs (control) with antibodies against KLF2 (Abcam, ab203591), H3K27Ac (Abcam, ab4729), and H3K4me3 (Abcam, ab1012). Antibody against goat IgG (Abcam, ab37373) was used as a negative control. The quantitative PCR analysis was performed with the selected primers from the transcriptional start sites of the ATG7 promoter regions (Fig. S1 ), and the primer sequences were described in Table- S2. Values obtained from the ChIP assay were normalized with background obtained from the precipitation with respective control antibodies. Percent (%) input was analyzed using the standard formula. Each experiment was performed in triplicate at least 3 times.
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