Proteins were extracted into a buffer with detergent, separated by electrophoresis in sodium dodecyl sulfate–polyacrylamide gels, and then blotted onto nitrocellulose membranes. The membranes were incubated at 4°C with antibodies against RUNX3 (ab135248, 1:1,000 dilution, Abcam) and GAPDH (sc-32233, 1:1,000 dilution, Santa Cruz Biotechnology). After washing, the membranes were incubated with a secondary Mouse IgG antibody (sc-2005, 1:2,000 dilution; Santa Cruz Biotechnology) for 2 h at room temperature. Blots were visualized using electrochemiluminescence (Amersham Pharmacia Biotech). The molecular weights of RUNX3 and E-cadherin were 44 and 120 kDa, respectively.
Ab135248
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab135248 is a laboratory reagent for use in scientific research. It is a polyclonal antibody that can be used to detect a specific protein target. The product information does not provide further details about the intended use or functionality of this reagent.
Automatically generated - may contain errors
Lab products found in correlation
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
A32731, by Thermo Fisher Scientific (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab76541, by Abcam (1 mentions)
Bca kit, by Boster Bio (1 mentions)
Ab32572, by Abcam (1 mentions)
Goat serum, by Solarbio (1 mentions)
Zli 9017, by ZSGB-BIO (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab28815, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab32101, by Abcam (1 mentions)
Enhanced chemiluminescence ecl kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Bca reagent kit, by Beyotime (1 mentions)
Ab56416, by Abcam (1 mentions)
5 protocols using ab135248
1
Western Blot Analysis of RUNX3 and GAPDH
2
Western Blot Analysis of Gastric Cancer Cells
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MKN7 and TMK1 cells were inoculated into 6-well plates, treated according to the above grouping, and cultured for 48 h. The cells were collected, lysed with RIPA (Solarbio, Beijing, China), and centrifuged at 3000 × g at 4°C for 15 min. The concentration of the extracted protein was determined using a BCA kit (Boster, Wuhan, China). Proteins (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electrically transferred to polyvinylidene fluoride (Millipore, USA) membranes. The membranes were blocked with TBST at room temperature for 1 h and incubated with anti-RUNX3 (1:1000, ab135248; Abcam), anti-CDX2 (1:10,000, ab76541; Abcam), anti-β-catenin (1:6000, ab32572; Abcam), anti-TCF-4 (1:1000, MA5-31923; Invitrogen, USA), anti-B-cell lymphoma-2 (Bcl-2; ab59348; Abcam, UK), and anti-Bcl-2-associated X protein (Bax; ab182733; Abcam) primary antibodies at 4°C overnight. After the membrane was fully washed with TBST, it was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (A32731) or goat anti-mouse IgG (A32723; Invitrogen, USA) secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence was used to detect the protein bands in a darkroom. GAPDH was used as an internal reference, and ImageJ software (NIH, USA) was used to analyze the grayscale changes.
3
Immunohistochemical Analysis of Rat Aortic Tissue
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The paraffin-embedded rat aortic tissues were sliced and the sections were routinely dehydrated with gradient ethanol and next immersed in potassium citrate at 90 °C for 10 min followed by antigen retrieval. The sections were washed with PBS three times and next added with 3% H2O2 to inactivate endogenous peroxidase for 10 min. After blocked with goat serum (Solarbio, Beijing, China) for 20 min, the sections were incubated with anti-rabbit Cbl (1: 200, PA5-8292, Invitrogen, Carlsbad, CA, USA), anti-rabbit p-JAK2 (1: 2000, ab32101, Abcam, UK), anti-rabbit p-STAT4 (1: 100, ab28815, Abcam), and anti-mouse Runx3 (1: 500, ab135248, Abcam) overnight at 4 °C. The samples were then incubated with secondary goat anti-mouse immunoglobulin G (IgG) (ab150113, Abcam) or goat anti-rabbit IgG (ab150077, Abcam) for 1 h at room temperature. Finally, the sections were developed with diaminobenzidine (DAB; ZLI-9017, ZSGB-BIO, Beijing, China) and 4 sections of each sample were observed under microscope (DMI3000, Leica Biosystems, Shanghai, China) in three random fields, as positive cell percentage was calculated.
4
Western Blot Analysis of Protein Expression
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Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor (KeyGene Biotech, China). Proteins (30 μg) were segregated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then electrophoretically transferred the protein onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin (BSA) for 2 h, the PVDF membranes were incubated with specific primary antibodies in recommended dilution ratio at 4°C overnight. The primary antibodies used in this study included anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-lg, Proteintech, China), anti-DGCR8 antibody (1:1,000, 10996-1-AP, Proteintech, China), and rabbit anti-RUNX3 antibody (1:1,000, ab135248, Abcam). Subsequently, the PVDF membranes were incubated with secondary antibodies (BOSTER, China) for 2 h. The protein strips were visualized and detected using a chemiluminescence reagent [enhanced chemiluminescence (ECL)] kit (Beyotime, China).
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) containing proteinase inhibitor (PMSF, Beyotime). The protein concentrations were measured using a BCA reagent kit (Beyotime). The membranes were probed, and the protein bands were visualized as previously described.24 The primary antibodies used include the following: anti‐β‐actin (4967, 1: 1000, Cell Signaling), anti‐GAPDH (60004‐1, 1: 5000, Proteintech), anti‐LC3B (3868, 1: 500, Cell Signaling), anti‐p62 (ab56416, 1: 2000, Abcam), anti‐RUNX3 (ab135248, 1: 1000, Abcam) and anti‐SIRT1 (ab110304, 1: 5000, Abcam). Housekeeping gene β‐actin or GAPDH served as the internal control.
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