CYP2E1 and GFP protein expression in the CYP2E1 and control stable clones of HepG2 cells were confirmed using western blot analysis of the microsomes. Briefly, 25 µg microsomes prepared from CYP2E1 and GFP clones of HepG2 cells was resolved by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a precast Bio-Rad gel (Hercules, CA, USA), and then transferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% (w/v) BSA for 1 h at room temperature. Subsequently, the membranes were probed overnight (4 °C) with rabbit CYP2E1 primary antibody (1:2000 dilution, Abcam, ab28146) or mouse anti-GFP (1:1000 dilution, OriGene, TA150041). To detect CYP2E1 protein, the membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000 dilution, Ab6721) and StrepTactin-HRP conjugate for 1 h at room temperature. To detect GFP protein, the membrane was incubated with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:2500 dilution, Invitrogen, 62-6520) and StrepTactin-HRP conjugate for 1 h at room temperature. The bands were detected using the Bio-Rad ChemiDoc Imaging system.
Ta150041
Manufactured by OriGene
Sourced in United States
TA150041 is a specialized lab equipment designed for general laboratory use. It serves as a basic tool for various research and experimental applications. The core function of TA150041 is to provide a reliable and consistent platform for conducting scientific investigations and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ab32505, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Euroclone (1 mentions)
Anti stat3, by Merck Group (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab32538, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti fgfr1, by Abcam (1 mentions)
Non fat dried milk, by Euroclone (1 mentions)
Anti pstat3 d3a7, by Cell Signaling Technology (1 mentions)
Ab81283, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
G8795, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
T3952, by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
7 protocols using ta150041
1
Western Blot Analysis of CYP2E1 and GFP Expression
2
Western blot analysis of signaling proteins
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Cell pellets were resuspended and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol), complemented with protease and phosphatase inhibitors cocktail (ThermoScientific). Total proteins extracts concentrations were determined through Bradford assay (Bio-Rad). Cytosol and nucleus protein fractions were obtained as previously described [47 (link)].
After 1 h blocking with 5% non-fat dried milk (EuroClone) or bovine serum albumin (SERVA) in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-pFGFR1 (06-1433, Millipore), anti-FGFR1 (Abcam ab137765), anti-pSTAT3 (D3A7, Cell Signaling), anti-STAT3 (06596, Millipore), anti-pAKT1 (ab81283; Abcam), anti-AKT1 (ab32505; Abcam), anti-pERK1/2 (ab32538; Abcam), anti-ERK1/2 (ab17942; Abcam), anti t-GFP (TA150041, Origene) and β-actin (Sigma, A5441). After membrane incubation with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Immuno Reagents), the positive bands were visualized using the ECL kit SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) as previously shown [48 (link)].
After 1 h blocking with 5% non-fat dried milk (EuroClone) or bovine serum albumin (SERVA) in Tris-buffered saline with 0.1% Tween (TBS-T), membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used: anti-pFGFR1 (06-1433, Millipore), anti-FGFR1 (Abcam ab137765), anti-pSTAT3 (D3A7, Cell Signaling), anti-STAT3 (06596, Millipore), anti-pAKT1 (ab81283; Abcam), anti-AKT1 (ab32505; Abcam), anti-pERK1/2 (ab32538; Abcam), anti-ERK1/2 (ab17942; Abcam), anti t-GFP (TA150041, Origene) and β-actin (Sigma, A5441). After membrane incubation with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Immuno Reagents), the positive bands were visualized using the ECL kit SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) as previously shown [48 (link)].
3
Visualizing Treacle-GFP Localization
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The cells were fixed and stained 48 h after transfection. The locations of treacle‐GFP fusion proteins were confirmed by detection with the following: anti‐TCOF1 serum diluted to 1:1000 (ab65212, Abcam); mouse monoclonal turboGFP antibody, clone OTI2H8 GFP at 1:5000 (TA150041; Origene); and TP53 mouse monoclonal antibody, clone DO‐1 at 1:1000 (TA804804; Origene). Images were obtained with a laser scanning confocal microscope (FV‐1000, Olympus, Japan).
4
Immunoblotting Analysis of GRIK Receptors
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HEK-293 cells were homogenized in 500 μL lysis solution containing 100 mM Tris-HCl (pH = 9.0), 100 mM NaCl, 0.5% (v/v) Triton X-100, and a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). The homogenates were centrifuged at 13,000 rpm for 60 min at 4 °C, and the supernatants were stored at −80 °C until use. Protein concentrations were determined by the Coomassie protein assay (Pierce Biotechnology Inc., Rockford, IL, USA). Immunoblotting analysis was performed according to standard protocols with the following primary antibodies: rabbit anti-GRIK1 (ARG55272, arigo Biolaboratories Corp, Taiwan), rabbit anti-GRIK2 (tcua3837, Taiclone, Taiwan), rabbit anti-GRIK3 (A10701, ABclonal, Woburn, MA, USA), rabbit anti-GRIK4 (tcba3836, Taiclone), mouse anti-tGFP (TA150041, Origene), and mouse anti-GAPDH (G8795, Sigma-Aldrich, Saint Louis, MO, USA). Anti-mouse IgG and goat anti-rabbit IgG conjugated with horseradish peroxidase were used as the secondary antibodies. The chemiluminescence signal was visualized using an ECL detection system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The intensity of the immunoblot was assessed with NIH ImageJ software (http://rsb.info.nih.gov/nih-image/ , accessed on 1 April 2019).
5
Immunoblotting Analysis of Protein Targets
6
Immunohistochemical Staining of VDR and CYP24A1
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Staining of the paraffin‐embedded tissue was performed as previously described.18 Staining for the VDR was performed using VDR primary antibody (1:200, Sigma Aldrich SAB 4503071) in 0.05% PBST. Expression of CYP24A1 in xenografts were determined by using a turboGFP antibody (1:100, Origene TA150041) in 0.05% PBST to detect the tagged GFP. Whole tissue slide images were acquired using TissueFAXS (TissueGnostics, Vienna, Austria).
7
SARS-CoV-2 Nucleocapsid Protein Detection
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Antibody against tGFP (TA150041) was purchased from OriGene Technologies (Rockville, MD, USA), antibody against SARS-CoV-2 Nucleocapsid protein (40143-R019) was from Sino Biological (Beijing, China), and β-Actin antibody (A1978) was from Sigma-Aldrich (St. Louis, MO, USA). IRDye 680- or 800-labeled secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE, USA). E64-D (S7393) and remdesivir (S8932) were purchased from Selleck (Houston, TX, USA). E64-D and remdesivir were kept in DMSO at the concentration of 40 mg/mL and 10 mmol/L as stock solutions respectively.
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