Cells were lysed and sonicated in the buffer containing 1% Triton X-100, 10 mmol/L TrisHCl (pH 7.4) and proteases/phosphates inhibitors (Roche Diagnostics, USA), electrophoresed in 10% SDS-PAGE gel electrophoresis, and transferred to a polyvinylidene difluoride membrane (PVDF) probed with primary antibodies to KPNA2. Subsequently, the membrane was probed with horseradish–peroxidase conjugated secondary antibodies. Then the blots were detected and visualized by a chemiluminescence detection system (Pierce, Rockford, IL, USA). The primary antibodies were used: anti-KPNA2 (Abcam, Cambridge, MA, USA), anti-c-myc(Abcam, Cambridge, MA, USA), anti-histoneH3 (Boster, Pleasanton, CA, USA); anti-E2F1 (Abcam, Cambridge, MA, USA), and anti-GAPDH (Boster, Pleasanton, CA, USA).
Anti e2f1
Manufactured by Abcam
Sourced in United States, China
Anti-E2F1 is a lab equipment product that detects the E2F1 transcription factor. E2F1 is a key regulator of the cell cycle and plays a role in the control of cell proliferation and apoptosis. The Anti-E2F1 product can be used for applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (4 mentions)
Anti p rb, by Abcam (3 mentions)
Anti rb, by Abcam (3 mentions)
Anti c myc, by Abcam (2 mentions)
Protein inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Anti tubulin, by Abcam (2 mentions)
Anti v5, by Abcam (2 mentions)
Anti foxa1, by Abcam (2 mentions)
Sds gradient gels, by Bio-Rad (2 mentions)
Anti e2f1, by Cell Signaling Technology (2 mentions)
Anti rb, by Cell Signaling Technology (2 mentions)
Anti corest, by Abcam (2 mentions)
Anti lsd1, by Abcam (2 mentions)
Anti h3, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti flag, by Proteintech (2 mentions)
Aj1236b, by Thermo Fisher Scientific (2 mentions)
Anti gfp, by Proteintech (2 mentions)
Anti mcherry, by Proteintech (2 mentions)
21 protocols using anti e2f1
1
Western Blot Analysis of KPNA2 Expression
2
Immunoprecipitation and Western Blotting
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For immunoprecipitation (IP), cells were lysed in Triton lysis buffer supplemented with protein inhibitor cocktails (Thermo Fisher Scientific), followed by a brief sonication, and then lysates were immunoprecipitated with anti-E2F1 (Abcam), anti-LSD1 (Abcam), or anti-CoREST (Abcam) antibodies. For immunoblotting, proteins were separated on 4–15% SDS gradient gels (Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), and then probed with primary antibodies, including anti-Rb (Cell Signaling), anti-tubulin (Abcam), anti-LSD1 (Abcam), anti-CoREST (Abcam), anti-E2F1 (Cell Signaling), anti-GAPDH (Abcam), anti-V5 (Abcam), anti-H3 (Abcam) and anti-FOXA1 (Abcam).
3
Protein Expression Analysis of ADSCs
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ADSCs were scraped off the culture plates, washed with PBS, and lysed with RIPA lysis buffer (1 mM MgCl2, 10 mM Tris‐HCl pH 7.4, 1% Triton X‐100, 0.1% sodium dodecyl sulfate [SDS], and 1% Nonidet P40 cocktail). The proteins were separated with 10% SDS‐polyacrylamide gel electrophoresis, and transferred to cellulose membranes. The membranes were incubated overnight with the following primary antibodies: anti‐CDK6 (Abcam, UK), anti‐CDC25A (Abcam), anti‐pRb (Abcam), anti‐E2F1 (Abcam), and anti‐β actin (Bioss, China). They were then immunoblotted with an antirabbit horseradish peroxidase‐conjugated secondary antibody (Abcam). Immunoreactivity was visualized with enhanced chemiluminescence and analyzed with the Quantity One system.
4
Protein Extraction and Western Blot Analysis
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Total protein from frozen tissues or cells was extracted using a Total Protein Extraction Kit ((Beyotime). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Equal amount of protein was run by 10% SDS-PAGE and blotted on a PVDF membrane (Millipore). To block non-specific sites, the membranes were incubated in 5% dry milk in TBS-T saline (0.25 M Tris-HCl; pH 7.6, 0.19 M NaCl, 0.1% Tween 20) for 2 h, the membrane immunodetected with anti-IRF5 (Abcam), anti-E2F1 (Abcam), and anti-GAPDH antibody (Proteintech) at 4 °C overnight at a dilution of 1:2000–1:4000. Then membranes were washed three times with TBS-T and treated with goat anti-rabbit IgG (Proteintech). Signals of membranes were measured by chemiluminescence (ECL) system, scanned and analyzed by Image Lab Software (Bio-Rad)
5
Quantitative Protein Expression Analysis
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Cell protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, CA, USA). GAPDH antibody was used as a control. Anti-E2F1 (ab179445, 1:1000), -CDK4 (ab199728, 1:1000), -CDK6 (ab199728, 1:1000), -Ki67 (ab16667, 1:1000), -Bcl-2 (ab182858, 1:1000), -ILF3 (ab131004, 1:1000), -MAP2K6 (ab33866, 1:1000), and -GAPDH (ab9485, 1:2000) antibodies were purchased from Abcam. Anti -CDK6 (3136s, 1:1000), -P21 (2947s, 1:1000) were purchased from Cell Signaling Technology.
6
Characterization of DAPK and miRNA pathway
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HeLa and 293Tcells were cultured in MEDM plus 10% FBS. Both cells were determined to be free from mycoplasma contamination. The antibodies used were from different vendors: Anti-DAPK1 (Abcam, ab109382; ABclonal, A5741), Anti-DAPK2 (Abgent, AP7033A), Anti-DAPK3 (Abgent, AJ1236b; Thermal scientific, PA5–27700), Anti-ACTIN (PTGCN, 60008-I-Ig), Anti-E2F1 (Abcam, ab179445), Anti-GW182 (Bethyl, A302–329A), Anti-TNRC6B (Abnova), Anti-TNRC6C (Bethyl, A303–969A), Anti-Ago1 (Abcam, ab105104), Anti-Ago2 (Abnova, H00027161-M01), Anti-Ago3 (Proteintech, 19692-I-AP), Anti-Ago4 (CST, 6913S), Anti-CD99 (Proteintech, 60354-I-Ig; ABclonal, A2028), Anti-NDRG1 (Abcam, ab124698), Anti-SAFB (Abnova, H00006294-M04), Anti-FLAG (ABclonal, AE005), Anti-GFP (Proteintech, 66002-I-Ig), anti-Mcherry (ABclonal, AE002). Western blotting results were quantified by the ImageJ software.
7
Electrophoretic Mobility Shift Assay Protocol
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For EMSA, the Wt and Mut short oligonucleotide probes were synthesized by GenePharma (Shanghai, China). The probes were biotin end-labeled using the Biotin 3' End DNA Labeling Kit (Thermo Pierce, Massachusetts, USA). The labeled and unlabeled probes were annealed to double-stranded probe DNA. EMSA assays were carried out using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Massachusetts, USA). For the super shift assay, anti-E2F1 (Abcam, #ab288369, Cambridge, UK) antibody or IgG (Cell signaling technology, #3900, Boston, Massachusetts, USA) were added to the nuclear extracts. All mixtures were separated on 6% native polyacrylamide and then transferred onto positively charged nylon membranes (Amersham Pharmacia, Piscataway, NJ, USA). After crosslinking the DNA by UV, the biotin-labeled DNA was identified using chemiluminescence. Probe sequences used for EMSA were listed in Table S1 .
8
Characterization of DAPK and miRNA pathway
9
Immunohistochemical Scoring of MELK and E2F1
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Briefly, the primary antibodies anti-MELK (1:200, Sigma), anti-E2F1 (1:100, Abcam) and anti-Ki67 (1:1000, Abcam) were incubated with the slides first. Each tumor was assigned a score according to the intensity of the nuclear or cytoplasmic staining (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) and the proportion of stained tumor cells: (0) <5%, (1) 5–25%, (2) 25–50%, (3) 50–75%, and (4) 75–100%. The scoring was judged independently by two pathologists in a blinded manner. The final immunoreactive score was determined by multiplying the intensity scores by the proportions of stained cells, resulting in “−” for a score of 0, “+” for a score of 1–4, “++” for a score of 5–8, and “+++” for a score of 9–12. Tumors with scores ≥5 were defined as tumors with high expression of MELK (or E2F1), whereas the others were defined as tumors with low MELK (or E2F1) expression.
10
Immunoprecipitation and Western Blotting
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