Cells were lysed to extract the entire protein contained in a radioimmunoprecipitation assay buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS] containing 1×protease inhibitor buffer. Protein concentrations were determined using a BCA kit (Thermo Scientific, Shanghai, China). Equal amounts of protein lysates were fractionated by SDS-PAGE and transferred to nitrocellulose membranes (PALL, Shanghai, China). The membranes were blocked with 5% skimmed milk and then incubated with gentle shaking overnight at 4°C, with the primary antibody plus 5% bovine serum albumin in Tris-buffered saline with Tween (TBS-T: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The following primary antibodies are β-actin, GAPDH (1: 1,000; Cell Signaling Technology, Shanghai, China), cytokeratin 18 (1: 1,000; Abcam, Shanghai, China), and SV40T (1: 100; Santa Cruze, Shanghai, China). The horseradish peroxidase (HRP)-conjugated secondary antibodies are goat anti-rabbit IgG and horse anti-mouse IgG (1: 5,000; Cell Signaling Technology). The target bands were detected using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific).
Sv40t
Manufactured by Santa Cruz Biotechnology
Sourced in China
The SV40T is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a protein that functions as a large T antigen, which is derived from the Simian Virus 40 (SV40). The SV40T protein is commonly used in research applications as a tool for cellular immortalization and transformation.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Cytokeratin 18, by Abcam (1 mentions)
Horse anti mouse igg, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab108349, by Abcam (1 mentions)
Gfap mab360, by Merck Group (1 mentions)
Ab178846, by Abcam (1 mentions)
Mhcii, by BD (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Sp kit sp 9000, by ZSGB-BIO (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
G box imaging system, by Syngene (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg apc, by Bioss Antibodies (1 mentions)
Goat anti mouse igg, by Abbkine (1 mentions)
Dylight 594, by Abbkine (1 mentions)
Tcs sp8, by Leica (1 mentions)
8 protocols using sv40t
1
Protein Extraction and Western Blot Analysis
2
Immunostaining of Neurological Markers
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The following antibodies were used in this study: Iba1 and p16 (ab178846 and ab108349, respectively, abcam, Cambridge, MA, USA); CD68 (MCAA1957GA, Bio-Rad, Hercules, CA, USA); MHCII (#556999, BD Biosciences, San Jose, CA, USA); CD45 (YW62.3, Thermo Fisher Scientific, Waltham, MA, USA); SV40T and PCNA (Pab 101 and PC10, respectively, Santa Cruz Biotechnology, Dallas, TX, USA); GFAP (MAB360) (Millipore, Billerica, MA, USA); Alexa Fluor conjugated goat-anti mouse 488, goat-anti rabbit 594, and goat-anti-rat 594 (Thermo Fisher Scientific). PHF1 antibody (generously provided by Dr. Peter Davies, The Feinstein Institute for Medical Research, Manhasset, NY); 4G8 (aa17-24 Aβ, Biolegend, San Diego, CA); Aβ42 (C42, C-terminal human Aβ42, IBL-America, Minneapolis, MN)
3
Immunohistochemical Staining Protocol
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IHC staining protocol was carried out by using the IHC staining (SP Kit, SP-9001, ZSGB-Bio, China) as described [73 (link),74 ]. Briefly, the sections were deparaffinized and subjected to immunostaining with the primary antibodies against Krt10 (1:50 dilution; Bimake; Cat# A5266), Krt15 (1:50 dilution; Bimake; Cat# A5627), Involucrin (1:50 dilution; Bimake; Cat# A5788), SV40 T (1:50 dilution; Santa Cruz; Cat# sc-147). The Biotin labeled goat anti-mouse IgG (SP Kit, SP-9000, ZSGB-Bio, China) or goat anti-rabbit IgG (SP Kit, SP-9001, ZSGB-Bio, China) were used to visualize the presence of the proteins of interest. Hematoxylin was used to stain the nuclei. Sections incubated without primary antibodies were used negative controls. The results were recorded under a bright field microscope (Leica DM4B/Nikon 80i).
4
Western Blot Analysis of SV40 T Antigen
5
Immunofluorescence Staining Procedure
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The IF staining was carried out as previously reported [24 (link),25 (link),27 ,65 (link),66 ]. Briefly, cells seeded and treated in chamber slides were fixed with 4 % paraformaldehyde for 15 min at RT, treated with 0.5 % Triton X-100 for 20 min, and blocked with 5 % goat serum (1:10 dilution) for 20 min at RT, followed by incubating with primary antibodies against SV40 T (1:50 dilution; Santa Cruz; Cat# sc-147), Krt10 (1:50 dilution; Bimake; Cat# A5266), Krt14 (1:50 dilution; Bimake; Cat# A5434), and Krt15 (1:50 dilution; Bimake; Cat# A5627) overnight. After being washed, the cells were incubated with DyLight 594, goat anti-mouse IgG (1:200 dilution; Abbkine; Cat# A23410) or goat anti-rabbit IgG/APC (1:200 dilution; Bioss; Cat# bs-0295G-APC). The nuclei were counterstained with DAPI (10 μg/mL). Minus primary antibody or control IgG was used as a negative control. IF results were recorded by using a laser confocal microscope (Leica TCS SP8).
6
Western Blot Analysis of Cellular Proteins
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The whole cell protein extract was prepared using lysis buffer comprising 0.5 M LSBD (0.5 M NaCl, 50 mM Tris–HCl pH 7.9, 20 % glycerol, 1 % NP-40, 1 mM DTT), 0.3 % NP-40, 1× Protease Inhibitor Cocktail (Roche), 1 mM NaF, 10 mM Na3VO4, 1 mM PMSF, 0.125 μM okadaic acid. The protein concentration of extracts was measured using a Protein Assay reagent (Bio-Rad Laboratories). Proteins (50 μg) were separated by SDS PAGE, transferred to nitrocellulose membranes, and incubated with indicated antibodies. Antibodies used were as follows: c-MYC (N-262, rabbit; Santa Cruz, sc-764), SV40 T (Pab 108, mouse; Santa Cruz, sc-148), and β-actin (C-11, goat; Santa Cruz, sc-1615).
7
Western Blot Analysis of Cellular Proteins
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Cells were harvested after washing with ice-cold phosphate-buffered saline (PBS) and solubilized in a solution containing 8 M urea, 50 mM Tris HCl (pH 7.4), and 0.15 M β-mercaptoethanol. Proteins were fractionated on polyacrylamide gels in the presence of 0.1 M Tris, 0.1 M bicine, and 0.1% SDS. For Western blotting, proteins were electrophoretically transferred to nitrocellulose membranes before incubation with antibodies overnight at 4°C. Antibodies used in the study were antibodies to Tab182 (an antibody raised in rabbits against GST-Tab182 [C-terminal fragment]), MRE11, CNOT3, CNOT4, CNOT7, (all from GeneTex), CNOT1 (Proteintech), cullin 2, cyclin E1, RPA32 (Abcam), p53 (raised in rabbits), cullin 5, GAPDH, collagen IV, SV40T (Santa Cruz Biotechnology), and β-actin (Sigma-Aldrich). Rabbit antibodies against Ad5 hexon and Ad12 fiber proteins were gifts from Vivien Mautner and Paul Freimuth, respectively. A mouse monoclonal antibody against the Ad5 DBP was a gift from Pieter van der Vliet. Antibodies against Ad5E1A (M73), Ad12E1A (5DO2), Ad12E1B55K (XPH9), Ad5E1B55K (2A6), p53 (DO1), and HA (12CA5) were purified from monoclonal supernatants.
8
Western Blot Protein Analysis
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Cells were lysed to extract total protein in radioimmunoprecipitation assay buffer containing protease inhibitors. The protein concentrations were determined using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein lysates were fractionated by SDS-PAGE and transferred to nitrocellulose membranes (Pall, Shanghai, China). Membranes were blocked with 5% skim milk and then incubated with gentle shaking overnight at 4°C with the primary antibody plus 5% horse serum in Tris-buffered saline with Tween (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The following primary antibodies were used: SV40T (1:200; Santa Cruz, Shanghai, China). Horseradish peroxidaseconjugated secondary antibodies were horse anti-mouse IgG (1:5,000; CST, Shanghai, China). Target bands were detected using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific).
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