RIPA buffer was applied to extract total cellular protein. The concentration of the protein was quantified by BCA analysis. Then sodium dodecyl sulfate-polyacrylamide sodium gel electrophoresis (SDS-PAGE) and PVDF membrane (Millipore, Bedford, MA) were used to separate the protein. The PVDF membrane was blocked with 5% skim milk for 1 h and incubated overnight with anti-GAPDH (1:2000, ab8245, Abcam) and anti-TK1 (1:1000, ab76495, Abcam) antibody at 4°C. The next day, the membrane was washed and incubated with HRP-conjugated goat anti-rabbit IgG antibody at room temperature for 1 h. Visualization and photography were performed using immobilon western chemilum hrp substrate (WBKLS0100, Millipore).
Ab76495
Manufactured by Abcam
Sourced in United States
Ab76495 is a lab equipment product from Abcam. It is a general-purpose device used for conducting experiments in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Abcam (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab113642, by Abcam (1 mentions)
Ab13575, by Abcam (1 mentions)
Ab13585, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Ab21685, by Abcam (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Sc 11733, by Santa Cruz Biotechnology (1 mentions)
Halt protease inhibitor cocktail 100, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Imagequant las 3000 mini system, by GE Healthcare (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Ab69120, by Abcam (1 mentions)
Nupage novex bis tris 4 14 gel, by Thermo Fisher Scientific (1 mentions)
Qdot 625 conjugate kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab76495
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Apoptosis Markers
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The protein levels of TK, VP3, Grp78, and HIF-1α as well as p21, p53, APC1, cytochrome C,
and caspase-3 were determined by Western blotting. Briefly, total proteins were extracted
from NPC cells, and the proteins were quantified with protein assay reagent from Bio-Rad
(Hercules, California). Proteins were then loaded on 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride or
polyvinylidene difluoride membranes. The membranes were subsequently blocked using 5%
nonfat milk at room temperature for 1 hour. And then membranes were probed with the
specific primary antibodies (all purchased from Abcam [Cambridge, MA, USA]) against TK
(anti-thymidine kinase 1 antibody, ab76495, 1/5000), VP3 (anti-VP3 antibody, ab193612,
1/5000), Grp78 (anti-Grp78 antibody ab21685, 1/500), HIF-1α (anti-HIF-1α antibody,
ab113642,1/500), p21 (anti-p21 antibody, ab109520, 1/1000), p53 (anti-p53 antibody, ab26,
1/500), APC1 (anti-Apc1 antibody, ab133397, 1/500), cytochrome C (anti-cytochrome C
antibody, ab13575, 1/500), caspase-3 (anti-caspase-3 antibody, ab13585, 1/500) at 4°C
overnight and subsequently incubated with corresponding secondary antibody at 37°C for 45
minutes. Protein bands were scanned with the Pierce ECL Western Blotting Substrate
(Pierce, Shanghai, China). β-Actin was served as an internal control.
and caspase-3 were determined by Western blotting. Briefly, total proteins were extracted
from NPC cells, and the proteins were quantified with protein assay reagent from Bio-Rad
(Hercules, California). Proteins were then loaded on 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride or
polyvinylidene difluoride membranes. The membranes were subsequently blocked using 5%
nonfat milk at room temperature for 1 hour. And then membranes were probed with the
specific primary antibodies (all purchased from Abcam [Cambridge, MA, USA]) against TK
(anti-thymidine kinase 1 antibody, ab76495, 1/5000), VP3 (anti-VP3 antibody, ab193612,
1/5000), Grp78 (anti-Grp78 antibody ab21685, 1/500), HIF-1α (anti-HIF-1α antibody,
ab113642,1/500), p21 (anti-p21 antibody, ab109520, 1/1000), p53 (anti-p53 antibody, ab26,
1/500), APC1 (anti-Apc1 antibody, ab133397, 1/500), cytochrome C (anti-cytochrome C
antibody, ab13575, 1/500), caspase-3 (anti-caspase-3 antibody, ab13585, 1/500) at 4°C
overnight and subsequently incubated with corresponding secondary antibody at 37°C for 45
minutes. Protein bands were scanned with the Pierce ECL Western Blotting Substrate
(Pierce, Shanghai, China). β-Actin was served as an internal control.
3
Knockdown of TK1 and RRM1 Confirmed
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The TK1 and RRM1 knockdown by specific siRNA were confirmed by western blot analysis. One, two and three days after siRNA transfection, LoVo cells were washed with PBS and 200 μL of lysis buffer was used to harvest the cells for Western blot analysis. Cell lysate proteins (20 μg) were used for immunoblot analyses using antibodies against RRM1 (sc-11733; Santa Cruz Biotechnology, Heidelberg, Germany) and TK1 (ab76495; Abcam, Paris, France).
4
Validating TK1 siRNA Knockdown in MDA-MB-231 Cells
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For our TK1 siRNA experiments we used the validated TK1 siRNA s14160 Silencer® Select (ThermoFisher Scientific, Waltham, MA). The Silencer™ GAPDH (Cat. No. 4390849) siRNA was used as positive control and the Silencer™ siRNA control No. 1 (Cat. No.4390843) was used as negative control. A total of 2.5 × 105 MDA-MB-231 cells were seeded in a 6 well plate and incubated overnight at 37 º C and 5% CO2. Cells were then transfected using 7.5 μl of lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) and 30 pmol of each corresponding siRNA following the manufacturer's protocol. After 24 h transfection was repeated. Cells were harvested after 24 h after second transfection for subsequent experiments and cell lysate preparations. Cell lysates were prepared using NP-40 cell lysis buffer with Halt™ protease inhibitor cocktail (100×) (ThermoFisher Scientific, Waltham, MA) and 1 mM of phenylmethylsulfonyl fluoride (Millipore SIGMA, Burlington, MA). Lysates were cleared by centrifugation and flash frozen with liquid nitrogen and stored at − 80 °C. The cell lysates were then analyzed with Western blot comparing cell lysates treated with TK1 siRNA with the GAPDH siRNA and siRNA negative controls for six custom TK1 antibodies and the commercial TK1 antibody Ab76495 (Abcam, Cambridge, UK).
5
Western Blot Analysis of TK1 and ENT1
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Cell pellets were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors cocktails (Nacalai Tesque, Kyoto, Japan), and incubated for 30 minutes on ice. The supernatant was cleared by centrifugation at 15000 × g and 5° C for 15 minutes. Because ENT1 is localized to the cell membrane [30 (link)], lysates were also prepared using Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (10 µg/lane for TK1 and 0.2 µg/lane for ENT1) were resolved by SDS-PAGE and analyzed by western blot using antibodies against TK1 (ab76495, Abcam, Cambridge, MA, USA) and ENT1 (sc377283, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were visualized on an ImageQuant LAS 3000 Mini system (GE Healthcare UK Ltd.).
6
Quantitative Western Blot Analysis
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Cells were plated overnight at a density of 1 × 106 cells per well on a 6-well plate. Cells were washed with PBS and lysed with 150 μL RIPA buffer containing protease and phosphatase inhibitor cocktail. The supernatants were collected by centrifuge at 14,000 g × 10 min at 4°C and stored at −80°C. The total protein content in lysates was determined by enhanced BCA protein assay kit (Beyotime Institute of Biotechnology, China). Electrophoresis was carried out on NuPAGE Novex Bis-Tris 4-14% gel (Invitrogen, USA) under the reduced condition with 5 μg of proteins per lane. The membrane was incubated with rabbit anti-TK1 monoclonal (ab76495, Abcam, USA) or anti-TYMP polyclonal antibody (ab69120, Abcam, USA) and mouse GAPDH antibody (Invitrogen, USA). Targeted proteins were visualized with Qdot 625 conjugate kit (Invitrogen, USA). Gel images were captured with ZF-258 Gel Imaging System (Shanghai Jiapeng Scientific Co. Ltd, China) under illuminating light of 350 nm wavelength.
7
Western Blot Analysis of Tumor Proteins
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Tumor tissue was lysed in radioimmunoprecipitation assay buffer with a Precellys 24 homogenizer/lysing kit (1.4 mm ceramic beads; Bertin Instruments). Polyacrylamide gel electrophoresis was performed with 4%-15% Mini-Protean TGX Precast Gels (BioRad), 15-well. After transfer to a Trans-Blot Turbo Midi Nitrocellulose membrane (Bio-Rad), proteins were probed overnight at 4°C with primary antibodies against TK1 (1:5,000; ab76495 [Abcam]), TS (1:1,000; 3766S [Cell Signaling Technologies]), TP (1:2,000; 12383-1AP [Acris]), or actin (1:10,000; ab6276 [Abcam]). Goat anti-rabbit (1:10,000; sc-2004 [Santa Cruz]) and goat anti-mouse (1:10,000; sc-2005 [Santa Cruz]) were used as secondary antibodies. Signals were then revealed with Amersham ECL Western blotting detection reagents and Amersham Hyperfilm ECL (both GE Healthcare). ImageJ was used for quantification via densitometry, and protein levels are expressed relative to an actin loading control.
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