Chondrocyte pellets were dissolved in citrate-EDTA buffer (55 mM/5 mM; pH 6.8) for 10 min at 37 °C and centrifuged at 10 K rpm for 15 s. Then, the cell pellet was washed five times with PBS and resuspended in 200 μL of SDS-NuPAGE buffer for NuPAGE gel (Invitrogen, Grand Island, NY, USA) running. Immunoblotting was performed using monoclonal antibodies against Type Ⅰ collagen, Type Ⅱ collagen (1:1000 dilution; EMD Millipore, Billerica, MA, USA), MMP-13 (Abcam, Cambridge, MA, USA) TGF-β (1:1000 mg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and PPAR-γ (Abcam, Cambridge, MA, USA). The loading control, β-actin, will be detected using anti-β-actin antibody (1:10,000 dilution; Abcam, Cambridge, MA, USA). Secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for TGF-β 1:20,000 dilution and mouse for type Ⅱ collagen and β-actin, 1:5000 dilution) was used. Proteins separated in the gel and the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm which was enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).
Amersham hyperfilm
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Germany
Amersham Hyperfilm is a high-performance X-ray film for use in autoradiography and chemiluminescence detection. It is designed to provide high sensitivity and excellent image quality for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blot substrate reagents, by GE Healthcare (6 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (4 mentions)
Enhanced chemiluminescence substrate, by GE Healthcare (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Anti β actin antibody, by Abcam (2 mentions)
Gahk20, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (2 mentions)
Complete mini protease inhibitor cocktail, by Roche (2 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Ecl western blot substrate buffer, by GE Healthcare (2 mentions)
Structurix x ray film processor, by GE Healthcare (2 mentions)
Clarity ecl substrate, by Bio-Rad (2 mentions)
89 protocols using amersham hyperfilm
1
Chondrocyte Protein Expression Analysis
2
SDS-PAGE and Western Blot Analysis of RBD Proteins
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The purified RBD proteins were analyzed by SDS-PAGE and Western blot as previously described.27 (link),29 (link) Briefly, proteins were separated by 10% Tris-glycine SDS-PAGE and stained with Coomassie brilliant blue or transferred to nitrocellulose membranes. The blots were blocked with 5% fat-free milk in PBS containing 0.5% Tween-20 (PBST) for 2 h at 37 °C and further incubated with SARS-CoV RBD-specific polyclonal antibody (mouse sera, 1:3,000),30 (link) or MERS-CoV RBD-specific antibody (mouse sera, 1:3,000),31 (link) overnight at 4 °C. The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature and then visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).
3
Western Blot Analysis of Protein Expression
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Cells were harvested by trypsinization and pelleted by centrifugation at 1000g for 2 min at 4°C. Cell pellets were resuspended in 500 µL lysis buffer (100 mM KCl, 0.1 mM EDTA, 20 mM HEPES-KOH pH 7.6, 0.4% NP-40, 10% glycerol, 1 mM DTT, complete mini EDTA-free protease inhibitors [Roche]) and clarified at 21,000g for 5 min at 4°C. A total of 250 µL supernatant was mixed with 20 µL 4× Bolt LDS sample buffer (Thermo Fisher Scientific), 8 µL 10× Bolt sample reducing agent (Invitrogen) and proteins were denatured at 75°C for 10 min. Protein samples were loaded into Bolt 4%–12% Bis-TRIS Plus gels (Thermo Fisher Scientific) and run at 160 V for ∼1 h. The gel was transferred onto an Amersham Hybond PVDF membrane (GE Healthcare), according to manufacturer's instructions. Primary antibodies were added at 1:10,000 concentration for α-V5 antibodies (Cat #V8012, Sigma-Aldrich), 1: 10,000 for α-puromycin antibodies (Cat #3RH11, Cedarlane), and 1: 5000 for α-tubulin antibodies (Cat #T5168 Sigma-Aldrich). α-mouse secondary antibody (Cat #7076, New England Biolabs) was used at 1:10,000. Blots were imaged using ECL Prime Western Blotting Detection Reagent (GE Healthcare) and exposed on Amersham Hyperfilm (GE Healthcare).
4
Quantifying Brain Glucose Utilization
5
Western Blot Protein Detection
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Proteins were separated by SDS–PAGE and subsequently transferred to nitrocellulose membranes (Amersham Bioscience) using a semi-dry apparatus (Trans-Blot SD, Bio-Rad). Membranes were blocked in 5% dry milk (Marvel) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated with primary antibodies (Table S4 ), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Table S4 ) and detection with enhanced chemiluminescence reagents (Amersham Bioscience) using Amersham hyperfilm (GE Healthcare) or the G:Box Chemi (Syngene).
6
Western Blot Analysis of Lipid Metabolism Proteins
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Tissue samples were lysed in RIPA buffer supplemented with complete mini protease inhibitor cocktail (Roche) and 50 mM Na3VO4 using a TissueLyzer (Qiagen). 20 µg protein was separated on 10% Tris–glycine gels. Transfer to a nitrocellulose membrane (GE) was performed in a wet blotting system for 2 h at 400 mA. Membranes were then blocked for 1 h in 5% milk in TBS-Tween (TBST) and after rinsing with TBST, membranes were incubated in the respective primary antibody overnight at 4 °C. Primary antibodies and dilutions were: CD36 (Novus Biologicals, 1:1000); LRP1 (Abgent, 1:25,000); LPL (Kind gift from Stefan K. Nilsson, 1:1000), SCD1 (Santa Cruz, 1:250), LPL (Cell Signaling, 1:1000). All primary antibodies were diluted in 5% BSA in TBS-T. After incubation, the membranes were washed 3 times 10 min in TBST and incubated for 1 h with HRP-coupled secondary antibodies (all from Jackson, all 1:5000 in 5% milk in TBS-T). After 3 additional washes in TBST, detection was performed using Amersham ECL Prime Western Blotting Detection Reagent (GE) and Amersham Hyperfilm (GE) autoradiography films. The films were developed using Kodak Developer and Fixer.
7
Western Blot Analysis of EGFR
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Fibroblasts lysates were prepared in RIPA buffer (R0278 Sigma) with complete protease inhibitor cocktail (Roche). Protein concentration in each sample was determined with Bradford protein assay and equal quantity of protein was loaded in Mini-PROTEAN 4–20% Tris-Glycine SDS-PAGE (Biorad) and transferred on nitrocellulose membranes (Santa Cruz). After incubation in TBS/non-fat milk 5% for 1 h, membranes were incubated in anti-EGF-R (ab52894, Abcam, 1/1000) or anti-GAPDH (CB1001, Millipore, 1/6000) diluted in TBS/Tween 0.1%/BSA 5% overnight at 4 °C. Membranes incubated in horseradish peroxidase conjugated secondary antibodies anti-rabbit (1:5000, 31,460, Thermofisher Scientific) or anti-mouse (1:5000, 31,430, Thermofisher Scientific) diluted in TBS/Tween 0.1% for 1 h, incubated in ECL substrate (34,580, Thermofisher Scientific) and developed on ECL films (Amersham Hyperfilm, GE). EGF-R level was normalized to GAPDH level in each condition.
8
Western Blot Analysis of ATP5A and Actin
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The pellet or supernatant from the previous centrifugation were mix NuPAGE LDS sample buffer (Thermo Fisher #NP0007) and heated at 70°C for 10 min. Samples were loaded onto NuPAGE 4% to 12% Bis-Tris Protein Gels (Thermo Fisher #NP0323BOX) and run with NuPAGE MES SDS running buffer (Thermo Fisher #NP0002). After semi-dry transfer, PVDF membrane (Millipore, Burlington, Massachusetts, USA, #IPVH00010) was blocked with 5% nonfat milk, and probed with anti-ATP5A (Abcam, Cambridge, United Kingdom, ab14748) or anti-actin (Abcam #ab3280) primary antibodies. The membrane was developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher #34096) and visualized by Amersham Hyperfilm (GE Healthcare, Chicago, Illinois, USA, #28906845).
9
Western Blot Analysis of PD-L1 and HIF-1α
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The cells were collected in PierceTM RIPA buffer (89901, Thermo Fisher Scientific) supplemented with 3% phosphatase and protease inhibitors (78442, Thermo Fisher Scientific) on ice and incubated for 30 min. Protein concentration was measured using the PierceTM BCA Protein Assay Kit (23225, Thermo Fisher Scientific) on a Nanodrop 2000 Spectrophotometer. Protein lysates (20 μg) were separated by electrophoresis on 4-20% Mini-PROTEAN@ TGXTM Precast Gel (#4561093, Bio-Rad) and transferred to a nitrocellulose membrane (1704271, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad).
Membranes were blocked with 5% non-fat milk solution (T145.1, ROTH) and then incubated for 1 h at RT employing either PD-L1 polyclonal antibody (PA5-20343, Invitrogen) (dilution 1:1000) or HIF-1α polyclonal antibody (PA1-16601, Invitrogen) (dilution 1:1000). β-actin (ab8227, Abcam) (dilution 1:2000) was used to standardize the loading. Polyclonal Goat Anti-Rabbit (P0448, Dako) HRP at a dilution 1:2000 was used as secondary antibody. The signals were detected utilizing Amersham ECL Western Blotting Detection Reagents (9838243, GE Healthcare, Freiburg, Germany) on Amersham Hyperfilm (28906836, GE Healthcare) on OPTIMAX X-Ray Film Processor (11701-9806-3716, PROTEC GmbH, Oberstenfeld, Germany). The ImageJ program (version 2, NIH, Washington, DC, USA) was used for densitometric analysis.
Membranes were blocked with 5% non-fat milk solution (T145.1, ROTH) and then incubated for 1 h at RT employing either PD-L1 polyclonal antibody (PA5-20343, Invitrogen) (dilution 1:1000) or HIF-1α polyclonal antibody (PA1-16601, Invitrogen) (dilution 1:1000). β-actin (ab8227, Abcam) (dilution 1:2000) was used to standardize the loading. Polyclonal Goat Anti-Rabbit (P0448, Dako) HRP at a dilution 1:2000 was used as secondary antibody. The signals were detected utilizing Amersham ECL Western Blotting Detection Reagents (9838243, GE Healthcare, Freiburg, Germany) on Amersham Hyperfilm (28906836, GE Healthcare) on OPTIMAX X-Ray Film Processor (11701-9806-3716, PROTEC GmbH, Oberstenfeld, Germany). The ImageJ program (version 2, NIH, Washington, DC, USA) was used for densitometric analysis.
10
PERV-C(5683) Viruses Structural Analysis
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Cell lysates from transfected ST-IOWA cells were analyzed for the presence of structural proteins by WB. Briefly, samples were subjected to SDS‐PAGE (10%) and transferred onto Immobilon‐P PVDF membranes (Merck Millipore, Germany). For functional analyses of HA-tagged PERV-C(5683) viruses, membrane was blocked with 0.1% TBS‐T containing 5% (w/v) skimmed milk (AppliChem, Germany) and proteins were detected with polyclonal rabbit Anti-PERV-C(498) antiserum (1:250) (peptide sequence: H2N-TGQRPPTQGPGPSSNI-COOH, manufactured by Eurogentec, Belgium) in 0.1% TBS‐T containing 5% (w/v) skimmed milk (AppliChem, Germany) at room temperature for 1 h. Infection assays of ST-IOWA/(PERV-C(5683) cells were analyzed for presence of Env and HA using the polyclonal rabbit anti-PERV-C(498) antiserum (1:250) and a mouse Anti-HA antibody (1 µg/mL) (GenScript, USA) in 0.1% TBS‐T containing 5% (w/v) skimmed milk (AppliChem, Germany) at room temperature for 1 h. Peroxidase-conjugated donkey anti-rabbit (Dianova, Germany); 1:15,000) or goat anti-mouse (Dianova, Germany); 1:10,000) secondary antibodies were incubated for 1 h in 0.1% TBS-T at room temperature. Anti‐beta‐actin (Abcam, USA); 1:10,000) was used as control. For detection, an Amersham Hyperfilm™ and ECL Western Blotting Detection Reagents (GE Healthcare, USA) were used.
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