RIPA buffer was used to extract total protein. Proteins were quantified using a BCA protein determination Kit (KeyGEN Biotech, Nanjing, China). 30 μg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h. The incubation of blots was conducted with antibodies at 4 °C overnight: Bax (ab32503, 1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (ab117115, 1:1000, Abcam), ILF3 (ab225626, 1:1000, Abcam), AURKA (ab108353, 1:1000, Abcam), E2F1 (ab4070, 1:500, Abcam), E-cadherin (ab231303, 1:1000, Abcam), Vimentin (ab92547, 1:1000, Abcam), PI3K (#3811, 1:1000, CST, Danvers, MA, USA), phosphorylated PI3K (p-PI3K, #4228, 1:1,000, CST), AKT (#9272, 1:1,000, CST), phosphorylated AKT (p-AKT, #9271, 1:1000, CST) or GAPDH (ab8245, ab9485, 1:5000, Abcam) and then with HRP-conjugated second antibody (#7074, 1:1000, CST). Protein bands were detected with ECL Plus reagent (Pharmacia, Piscataway, USA), and visualized using a Gel Imaging System. Bands were then quantified using ImageJ software (National Institutes of Health). The expression of GAPDH was used for data normalization.
Ab4070
Manufactured by Abcam
Sourced in United States
Ab4070 is a lab equipment product. It serves as a core function for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab117115, by Abcam (1 mentions)
Ab225626, by Abcam (1 mentions)
Ab108353, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bca protein determination kit, by Keygen Biotech (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab13537, by Abcam (1 mentions)
Sc 6954, by Santa Cruz Biotechnology (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab57653, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab2410, by Abcam (1 mentions)
Magna chip hisens chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Ab207732, by Abcam (1 mentions)
Ab134903, by Abcam (1 mentions)
Ab81299, by Abcam (1 mentions)
Ab170904, by Abcam (1 mentions)
7 protocols using ab4070
1
Western Blot Analysis of Apoptosis and EMT Markers
2
Immunoblotting Analysis of Cellular Proteins
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Two days after siRNA transfection, cells were harvested and lysed in 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM 2-glycerophosphate, 10 μg/mL leupeptin, 5 μg/mL aprotinin, 5 μg/mL pepstatin, and 20 mM microcystin-LR. Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology). Secondary antibodies used were: horseradish peroxidase-conjugated anti-mouse immunoglobulin G (1:2000 for BRCA1 and 1:10,000 for all other primary antibodies, 7076 S, Cell Signaling Technology) and anti-rabbit immunoglobulin G (1:16,000 for ZC3H18 and 1:10,000 for all other primary antibodies, 7074 S, Cell Signaling Technology).
3
Immunohistochemical Analysis of RCC Signaling
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Paraffin-embedded tumor tissue specimens were sliced into 5-μm-thick sections and mounted onto slides. Then, the slices were deparaffinized, rehydrated, subjected to antigen retrieval, and incubated with specific primary antibodies overnight at 4°C. Subsequently, the sections were incubated with secondary antibodies for 1 h after washing three times with PBS. After staining with diaminobenzidine (DAB), the sections were visualized under a microscope. In this study, the patient tumor sections were stained with phospho-p44/42 Erk1/2 (Thr202/Tyr204) (4370, Cell Signaling Technology) to assess the expression of the protein in RCC. The quantification of phospho-Erk1/2 was scored by the product of intensity and percentage of staining. The xenograft tumor sections were stained with anti-Ki67 (ab15580, Abcam), anti-E2F1 (ab4070, Abcam), anti-RRM1 (8637, Cell Signaling Technology), and RRM2 (ab57653, Abcam) to verify the molecular mechanisms detected in vitro.
4
ChIP Assay for E2F1-ASK1 Promoter Interaction
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ChIP assay was implemented to testify the interplay between E2F1 and ASK1 promoter via using the ChIP assay kit (KT101-01, gzscbio, Gunagzhou, China). Cells were first cross-linked in 4% paraformaldehyde and then sonicated into chromatin fragments of 200-1000-bp which were incubated with the antibodies against E2F1 (ab4070, Abcam, UK) and negative control IgG (ab6789, Abcam, UK). After that, magnetic beads were added to enrich RNA, and finally, the immunoprecipitated RNAs were extracted and purified.
5
ChIP Assay Protocol for E2F1 Binding
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ChIP assays were performed using a Pierce Agarose ChIP Kit according to the manufacturer’s protocol. PANC1 cells were crosslinked with 1% formaldehyde for 10 min at 37 °C and then incubated with anti-E2F1 antibodies (Abcam, ab4070) and anti-Rabbit IgG (Abcam, ab2410). Bound DNA fragments were subjected to RT-PCR using specific primers (Table 2 ).
Correlations between Linc00337 and key clinicopathological parameters
| Variable | Linc00337 (n = 18) | |||
|---|---|---|---|---|
| unchanged | Up-regulated | Chi-square P value | ||
| Age | ≤50 years | 1 | 8 | 0.527 |
| > 50 years | 2 | 7 | ||
| Gender | Female2 | 0 | 3 | 0.396 |
| Male1 | 3 | 12 | ||
| Grade | I | 2 | 0 | 0.003* |
| II and III | 1 | 15 | ||
| Tumor size | ≤ 2 cm | 2 | 2 | 0.043* |
| > 2 cm | 1 | 13 | ||
| TNM stage | T1 | 1 | 0 | 0.05* |
| T2 | 2 | 10 | ||
| T3-T4 | 0 | 5 | ||
*p ≤ 0.05
6
Chromatin Immunoprecipitation Assay for RRM1 and RRM2 Promoters
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ChIP assay was performed using Magna ChIP HiSens Chromatin Immunoprecipitation Kit (17-10460, Merck Millipore), according to the manufacturer’s instructions. The DNA fragments isolated in the complex with the target protein were identified by qPCR using primers for RRM1 and RRM2 promoters. The antibody used for ChIP was anti-E2F1 (ab4070, Abcam). The sequences of primers used for qPCR were as follows:
RRM1: 5′-GTTCCCGCCGGTTAGGTTTT-3′ and 5′-CTGCTCTCCTGCTACCATGT-3′, and
RRM2: 5′-TTTGGAAGTCGCGCTAACCT-3′ and 5′-GCGCGTTTTGACATCTGCAT-3′.
7
Western Blot Analysis of Stem Cell Markers
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Protein was extracted from cells with a protein lysis buffer (Beyotime). Total proteins were quantitated with a BCA assay kit (Pierce, Rockford, IL, USA), followed by separation with 10% SDS-PAGE gels. Subsequently, the proteins were removed from gel to PVDF membrane and then blocked with 5% milk for 1 h. After incubation with primary antibody overnight at 4 °C, the membranes were incubated by HRP-conjugated secondary antibody for 2 h. Lastly, the blots were measured by ECL substrate. The primary antibodies included: anti-CD133 (130–092–395, 1:100, Miltenyi Biotec), anti-CD151 (SAB1402716, 1:50, Sigma), anti-E2F1 (ab4070, 1:500; Abcam), anti-MDR1 (ab170904, 1:100, Abcam), anti-BCRP1 (ab207732, 1:100, Abcam), anti-γ-H2AX (ab81299, 1:400; Abcam), anti-H2AX (ab11175, 1:200, Abcam), anti-AKT (#9272, 1:1000, Cell Signaling, Danvers, MA, USA), anti-p-AKT (#9271, 1:1000, Cell Signaling), anti-mTOR (ab134903, 1:100, Abcam), anti-p-mTOR (#2971, 1:100, Cell Signaling), anti-cyclinD1 (#2922, 1:1000, Cell Signaling) and anti-β-actin (A1978, 1:6000, Sigma).
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