Total proteins were extracted from tissues and cells. Anti-GAPDH antibody (1:5000, A5441, Sigma), anti-CRIF1 (1:2000, 16260-1-AP, Proteintech), anti-cyclin D1 (1:2000, ab137875, Abcam), anti-cyclin E1 (1:1000, ab52189, Abcam), anti-CDK4 (1:1000, ab137675, Abcam), anti-CDK6 (1:1000, ab151247, Abcam), anti-MMP3 (1:1000, ab52915, Abcam), anti-TGF-β1 (1:500, ab9758, Abcam), anti-TGF-β2 (1:100, ab167655, Abcam), TGF-β receptor 1 (1:1000, ab155258, Abcam), anti-Smad2 (1:1000, 3122, CST), anti-Smad3 (1:1000, 9513, CST), anti-p-Smad2 (1:1000, 3108, CST), anti-p-Smad3 (1:1000, 9520, CST), anti-E-Cadherin (1:2000, ab133597, Abcam), and anti-N-Cadherin (1:1000, ab19348, Abcam) antibodies were used. Immunodetection was performed using an EZ-ECL chemiluminescence detection kit (BeitHaemek, Israel).
Ab151247
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab151247 is a lab equipment product offered by Abcam. It is a device designed for specific laboratory applications. The core function of this product is to perform a particular task or measurement required in a research or analytical setting. No further details on the intended use or performance specifications are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (5 mentions)
Ab134175, by Abcam (4 mentions)
Ab37168, by Abcam (4 mentions)
Ab137675, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab137875, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
A0208, by Beyotime (2 mentions)
Ab109526, by Abcam (2 mentions)
Ab76125, by Abcam (2 mentions)
Ab40776, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Ab16663, by Abcam (2 mentions)
Ab196495, by Abcam (2 mentions)
Ap0063, by Bioworld Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ab52915, by Abcam (1 mentions)
Ab133597, by Abcam (1 mentions)
Ab19348, by Abcam (1 mentions)
21 protocols using ab151247
1
Comprehensive Protein Analysis in Cells
2
Protein Expression Analysis in HaCaT Cells
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After exposure or not to LIPUS, HaCaT cells were lysed in RIPA buffer (Beyotime, China) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF; Beyotime). The whole lysates were centrifuged at 12,000 g for 10 min at 4°C, and the protein levels in the supernatants were quantified using the BCA™ Protein Assay Kit (Pierce, USA). Proteins were loaded onto SDS-PAGE gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Then, PVDF membranes were blocked with 5% bovine serum albumin (BSA; Solarbio, China), and incubated overnight at 4°C with specific primary antibodies for cyclin D1 (ab134175), cyclin-dependent kinase (CDK) 6 (ab151247), CDK4 (ab137675), vascular endothelial growth factor (VEGF; ab150766), matrix metalloproteinase (MMP) 2 (ab97779), MMP-9 (ab137867), PI3K (ab180967), phospho (p)-PI3K (ab182651), β-actin (ab8227, all Abcam), AKT (#9272), p-AKT (#9271), JNK (#9252), and p-JNK (#9251, all Cell Signaling Technology, USA). After rinsing, PVDF membranes were incubated with HRP-conjugated secondary antibody (goat anti-rabbit, ab205718, Abcam) at room temperature. An enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA) was used to detect the target proteins. Protein levels were quantified by ImageJ software (National Institutes of Health, USA).
3
Western Blot Analysis of CDK6 Expression
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SiHa cells were harvested at 24 h post-transfection and cells were counted. Cell pellets containing 105 SiHa cells were resuspended in 1 mL RIPA solution (Cell Signaling Technology) to extract total proteins. Concentrations of total proteins were measured using BCA assay (Cell Signaling Technology). In a 95°C incubator, protein samples were denatured for 10 min, followed by electrophoresis using 12% SDS-PAGE gel. After that, proteins were transfected to PVDF membranes and blocking was performed in 5% non-fat milk (PBS) at room temperature for 1 h. GAPDH (1:1800, ab37168, Abcam) and CDK6 (1:1800, ab151247, Abcam) rabbit polyclonal primary antibodies were used to incubate with the membranes at 4°C for 18 h, followed by incubation with HRP (IgG) (1:1800; ab6721; Abcam) goat anti-rabbit secondary antibody at 24°C for 2 h. ECL Western Blotting Substrate (Promega Corporation) was dropped onto the membranes and MYECL™ Imager (Bio-Rad) was used to detect signals. Image J v1.46 software was used for data normalizations.
4
Cervical Cancer Biomarker Expression Analysis
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Fresh cervical cancer tissues were acquired from 50 patients at Nanjing Maternity and Child Health Care Hospital from June 2017 to December 2019. Moreover, the healthy cervical tissues at the edge of the cervical tumor were obtained as the paracancerous tissue (control). Subsequently, the specimens were quickly snap-frozen in liquid nitrogen and transferred to −80°C for preservation. All the patients signed informed consent, and the hospital ethical committee approved this study (Permit number:2019-KY-007).
To determine whether a patient had a low or high expression of LINC00313, miR-4677-3p and CDK6. We analyzed the slices of the tumor samples by Immunohistochemistry or in situ hybridization (ISH). ISH probes (all purchased from GenePharma Inc., Shanghai, China) were employed for the expression detection of Lnc-RNA LINC00313 and miR-4677-3p. And, antibodies (ab151247, Abcam) were used for the expression detection of CDK6. The sample was identified as positive if more than 50% of total cells were positive.
To determine whether a patient had a low or high expression of LINC00313, miR-4677-3p and CDK6. We analyzed the slices of the tumor samples by Immunohistochemistry or in situ hybridization (ISH). ISH probes (all purchased from GenePharma Inc., Shanghai, China) were employed for the expression detection of Lnc-RNA LINC00313 and miR-4677-3p. And, antibodies (ab151247, Abcam) were used for the expression detection of CDK6. The sample was identified as positive if more than 50% of total cells were positive.
5
Western Blot Analysis of Cellular Markers
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The following antibodies were used in this study: anti-XTP1 (Abcam, USA, ab124182, 1:1,000), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Bioworld, USA, AP0063, 1:3,000), anti-rabbit immunoglobulin (IgG) (Beyotime, China, A0208, 1:3,000), anti-Ki-67 (Abcam, USA, Ab16667, 1:200), anti-rabbit IgG (Abcam, USA, ab6721, 1:400), anti-cluster of differentiation (CD)44 (Abcam, USA, ab157107, 1:2000), anti-cyclin-dependent kinase 6 (CDK6) (Abcam, USA, ab151247, 1:1,000), anti-ras homolog family member U (RHOU) (Abcam, USA, ab80315, 1:1,000), and anti-rabbit IgG (Beyotime, China, A0208, 1:3,000).
6
Immunoblotting of Cell Signaling Proteins
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Protein samples were boiled in SDS/β-mercaptoethanol sample buffer, and 20 μg of each sample was loaded into each lane of 4–12% polyacrylamide gels. After separation by electrophoresis, the proteins in the gels were transferred onto PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK). The membrane was incubated with rabbit anti-CDK6 polyclonal antibody (ab151247, Abcam, Cambridge, MA, USA) or rabbit anti-EZH2 monoclonal antibody (ab191080, Abcam, Cambridge, MA, USA) or rabbit anti-PDL1 polyclonal antibody (ab205921, Abcam, Cambridge, MA, USA) or rabbit anti-TAK1 polyclonal antibody (ab109526, Abcam, Cambridge, MA, USA) or rabbit anti-KRAS polyclonal antibody (ab180772, Abcam, Cambridge, MA, USA), or rabbit anti-FGF9 polyclonal antibody (ab71395, Abcam, Cambridge, MA, USA) or mouse anti-β-actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) over night at 4 °C. The specific protein-antibody complex was detected by using horseradish peroxidase conjugated goat anti-rabbit or rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit (Pierce, Appleton, WI, USA). The β-actin signal was used as a loading control.
7
Western Blot Analysis of Cell Signaling
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Whole cell lysates were extracted using either NETN buffer supplemented with protease inhibitor cocktail or direct lysis. Protein lysate was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore) for western blot analyses. Primary antibodies against CDK6, CDK4 [EPR2513Y], MAP3K7 (TAK1) [EPR5984] (1:1000, ab151247, ab68266, ab109526, Abcam), p-GSK3β (Ser9) (D3A4), β-catenin (D10A8), p44/42 MAPK, p-p44/42 MAPK, YAP (D8H1X), pYAP(Ser127) (D9W2I), SOX2 (D6D9), RPS6KA4 (MSK2) (D41A4) (1:1000, #9322 S, #8480 S, #9102 S, #9101 S, #14074 S, #13008, #3579, #3679, Cell Signalling Technology), GSK3β (clone 7) (1:1000, #610202, clone 7, BD Transduction Laboratories), HA (1C1D2) (1:1000, #66061-1-Ig-AP, Proteintech), OCT4 (C-10) (1:1000, sc5279, Santa Cruz) and β-ACTIN (AC-74) (1:5000, #A5316, Sigma Aldrich) were incubated at 4 °C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse or rabbit antibody (GE HealthCare). The signals were visualized using the enhanced chemiluminescence method. Blot images were quantified by densitometry using ImageJ software.
8
Western Blot Analysis of GSG2 and Apoptosis Proteins
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Western blot assay was applied to determine the GSG2 protein expression levels in shGSG2 HO-8910 cells. Cells were collected and lysed with 1 × cold lysis buffer. Protein was extracted with BCA protein detection kit (Cat. #23225, HyClone-Pierce). 10% SDS-PAGE was used for Western analysis. Blots were transferred to a PVDF membrane and incubated with blocking liquid for 1 h. GSG2 antibody (1:1000, Cat. #ab21686, Abcam), CCND1 antibody (1:3000, Cat. #ab134175, Abcam), CDK6 antibody (1:1000, Cat. #ab151247, Abcam), MAPK9 antibody (1:3000, Cat. #ab76125, Abcam), PIK3CA antibody (1:1000, Cat. #ab40776, Abcam), and internal standard GAPDH antibody (1:3000, Cat. #AP0063, Bioworld) were added overnight at 4°C. After incubated with secondary antibody HRP goat anti-rabbit IgG polyclonal (1:3000, Cat. # A0208, Beyotime) for 1 h at room temperature, the membrane was color developed using ECL-PLUS/Kit (Cat. #RPN2232, Amersham).
Human Apoptosis Antibody Array was purchased from Abcam (Cat. Ab134001) for detecting the expression of apoptosis-related proteins according to the manufacturer’s instructions.
Human Apoptosis Antibody Array was purchased from Abcam (Cat. Ab134001) for detecting the expression of apoptosis-related proteins according to the manufacturer’s instructions.
9
Western Blotting Procedure for Protein Analysis
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The protein used for Western blotting was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Guangzhou, P.R. China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The Western blot system was established using a Bio-Rad Bis-Tris Gel System according to the manufacturer’s instructions. Primary antibodies against YEATS4 (ab205018), GAPDH (ab128915), phosphorylated β-catenin (p-β-catenin, ab138378), β-catenin (ab6302), Bcl-2 (ab32124), Bax (ab77566), c-Myc (ab152146), CDK6 (ab151247), CDK4 (ab137818), and cyclin D1 (ab137875) (all from Abcam, Cambridge, MA, USA) were prepared in 5% blocking buffer. Primary antibodies were respectively incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibodies marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS System, and then 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was added to cover the membrane surface.
10
Western Blot Analysis of Cell Proteins
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After trypsinization, C-33A cells were counted and 105 cells were resuspended in 0.4 ml RIPA solution (RIBOBIO) to extract total proteins. All protein samples were quantified by performing a BCA assay. After protein denaturation in boiling water for 10 min, electrophoresis was performed using 10% -PAGE gel, followed by protein transfer to PVDF membranes. All membranes were incubated with 5% non-fat milk for 25 at 24 °C. Rabbit polyclonal primary antibodies of GAPDH (ab37168, 1:1100, Abcam) and CDK1 (ab151247, 1:1100, Abcam) were used to incubate the membranes for 1 h at 4 °C. HRP (IgG) goat anti-rabbit secondary antibody (ab6721, 1:900, Abcam) was used to further incubate with the membranes for 2 h at 24 °C. ECL (Sigma-Aldrich, USA) and ImageJ v1.46 software were used for signal development and normalization, respectively.
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