The human cervical cancer cell line CaSki was obtained from the American Type Culture Collection (Manassas, VA, USA) and resuscitated by the Cell Bank, CAS. CaSki cells were then cultured at 37°C in a humidified 5% CO2 atmosphere in Roswell Park Memorial Institute (RPMI)-1,640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). cis-Dichlorodiammineplatinum-II (CDDP) was purchased from Sigma-Aldrich (Merck KGaA). Rabbit monoclonal antibodies directed against p53 (2527), p21 Waf1/Cip1 (2947), p27 Kip1(3686s), apoptosis protease activating factor 1 (Apaf-1) (8969s), B cell lymphoma/leukemia-2 (Bcl-2) (9941), Bcl-2 associated X protein (Bax) (9942s), cleaved-caspase-9 (9501s), cleaved-caspase-3 (9664s) and GAPDH (2118) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse monoclonal antibody directed against human papilloma virus (HPV) E6 (sc-460) was provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal antibody against Ki-67 (ab16667) was also used (Abcam, Cambridge, UK).
Sc 460
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-460 is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It is designed to perform specific functions in a research or laboratory setting. The core function of the Sc-460 is to [description of core function, without extrapolation or interpretation].
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Ki67 ab16667, by Abcam (1 mentions)
Caski, by Thermo Fisher Scientific (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Caski, by American Type Culture Collection (1 mentions)
Iseq00010, by Merck Group (1 mentions)
P0028, by Beyotime (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab87656, by Abcam (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti dnmt1, by Abcam (1 mentions)
Ant β actin, by Abcam (1 mentions)
Anti e6, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
4 protocols using sc 460
1
CaSki Cell Line Cultivation and Protein Analysis
2
Western Blot Analysis of Cellular Signaling
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Proteins were extracted from the cells at the indicated times using SDS lysis buffer (P0013G, Beyotime, China). Nuclear and Cytoplasmic Extraction Reagents (P0028, Beyotime, China) were used to separate the cytoplasmic and nuclear extracts from cultured cells. The protein samples (30 μg) were subjected to 4–20% polyacrylamide gels (JC-PE022/JC-PE022R, Genshare biological, China) to be electrophoresed and transferred to 0.22 μm PVDF membranes (ISEQ00010, Merck Millipore, USA) at 90 V for 1.5 h. Membranes were blocked with 5% skim milk for about 1 h at room temperature and then incubated with primary antibodies against HPV 16 E6 (sc-460, Santa Cruz, USA; 1:200), IκBα (4814, CST, USA; 1:1000), p-IκBα (Ser 32/36) (9246, CST, USA; 1:1000), p65 (10745-1-AP, Proteintech, USA; 1:1000), p-p65 (Ser536) (3031, CST, USA; 1:1000), p-p65 (Ser468) (3039, CST, USA; 1:1000), H3 histone (4499, CST, USA; 1:1000), Akt (10176-2-AP, Proteintech, USA; 1:1000), p-Akt (Ser473) (4060, CST, USA; 1:1000), p-Akt (Thr308) (13038, CST, USA; 1:1000), GAPDH (Proteintech, USA; 1:10,000), and β-actin (3700, CST, USA; 1:1000) at 4 °C overnight. Thereafter, the membranes were probed with IR Dye-labeled secondary antibodies (800CW, LI-COR Bioscience, USA; 1:10,000) and the signals were observed using an Odyssey Infrared Imaging System (LI-COR Bioscience, USA).
3
Immunohistochemical Analysis of Cervical Cancer
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Cervical cancer samples were fixed in phosphate-buffered 10% formalin (pH 7.2), embedded in paraffin and cut into 4-µm sections. The sections were dewaxed in xylene, dehydrated in alcohol, and then incubated in 0.01 M sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated with 3% H2O2 for 30 min to block endogenous peroxidase activity, and with 10% milk at 37°C for 15 min to block non-specific binding of antibodies. Sections were then incubated with hWAPL, Oct4 or HPV E6 antibodies (1:100 dilution in PBS, cat. nos. sc-365189, sc-101534 and sc-460, respectively; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. This was followed by incubation with biotinylated secondary antibody (1:2,000, cat. no. sc-358919; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h and visualization using 3,3′-diaminobenzidine under a light microscope.
4
Western Blot Analysis of Protein Expression
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Total protein from cells were extracted by using RIPA buffer (50 mM TrisHCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40 and 0.1% SDS). Total protein concentration was measured by using the BCA protein assay (Beyotime, Shanghai, China). Samples containing 30 μg protein was loaded in each lane, separated on 10% SDS PAGE gel and then transferred onto nitrocellulose membranes for a conventional western blot analysis. Antibodies used include Anti-E6 (1:500, sc-460, Santa Cruz Biotechnology, Santa Cruz, CA, USA) anti-DNMT1 (1:2000, ab87656, Abcam, Cambridge, UK), anti-E-cadherin (1:1000, #3195, Cell Signaling, Danvers, MA, USA) and anti-N-cadherin (1:1000, #13116, Cell Signaling). β-actin served as loading control and was detected by using ant-β-actin (1:500, ab8229, Abcam). Membranes were washed and incubated with corresponding HRP-labeled secondary antibodies. Protein signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, lL, USA) and intensity of each band was quantified by ImageJ software. Results were shown by three independent experiments (n = 3).
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