Cell and tissue extracts were collected in radioimmunoprecipitation (RIPA) lysis buffer and protein concentrations were measured by Bradford assay (Bio-Rad) to equalize loading. Samples were boiled for 5 minutes in SDS buffer before being analyzed on polyacrylamide gels, transferred to nitrocellulose membrane (0.2 micron) and blocked for one hour in 5% milk 0.1% tween-20 tris buffered saline (TBST). All membranes were incubated overnight with primary antibody (GAPDH – Abcam # Ab8245; Stathmin-2 – Novus # NBP1-49461) immunoblots were then washed three times with TBST and probed with horseradish peroxidase conjugated secondary antibodies diluted 1:5,000–1:10,000 in 5% milk for 1 hour at room temperature before being exposed to films or imaged on a LICOR Fc imager.
Fc imager
Manufactured by LI COR
The Fc imager is a versatile imaging system designed for a range of fluorescence-based applications. It provides high-resolution imaging of fluorescent gels, blots, and microplates, enabling researchers to capture and analyze a variety of fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
β mercaptoethanol, by Merck Group (2 mentions)
Nbp1 49461, by Novus Biologicals (1 mentions)
Ab8245, by Novus Biologicals (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Myeloperoxidase mpo, by R&D Systems (1 mentions)
Ifn β, by PBL Assay Science (1 mentions)
Lds buffer, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ultrapure water, by Thermo Fisher Scientific (1 mentions)
Iblot 2 transfer device, by Thermo Fisher Scientific (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Precision plus dual color protein ladder, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Penta his antibody, by Qiagen (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Bio dot apparatus, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Proteome profiler human phospho rtk array kit, by R&D Systems (1 mentions)
7 protocols using fc imager
1
Western Blot Protein Detection Protocol
2
Quantification of VGL4-TEAD1 Interaction
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Synthetic biotinylated VGL4 was immobilized on 25 µL of streptavidin magnetic beads (#1420, New England Biolabs) for 1 h at room temperature. Beads were then extensively washed with the incubation buffer (300 mM NaCl, 25 mM Tris (pH 8.8), 2 mM DTT, 2% glycerol). Purified GST-hTEAD1 (1 µM) was incubated with the required amount of peptide (50 and 200 µM) in incubation buffer for 30 min at room temperature; then immobilized VGL4 was added to the protein/peptide complex (final volume 200 µL) and incubated for 2.5 h at room temperature. Beads were then extensively washed with the incubation buffer followed by protein elution after boiling the beads at 95 °C for 10 min in 25 µL of SDS loading buffer. SDS PAGE was performed using standard methods and the gel was visualized after Coomassie staining with a Li-COR Fc Imager.
3
Analyzing Small RNA via Northern Blot
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Northern blot analysis was modified from methods previously described [59 (link)]. Denaturing polyacrylamide gels (15%) containing 7M urea was used to separate small RNA. Equivalent loading and RNA integrity were confirmed by gel imaging of the ribosomal RNA using a Licor FC imager. Blots were probed with the 5’-biotinylated RNA probe: tRF5-Glu probe (Supplementary Table 3 ), and the microRNA marker miR -21 probe was used for size analysis (New England Biolabs, Ipswich, MA).
4
Tumor Homogenate Analysis for Viral Titer and Inflammatory Markers
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Tumor homogenate was prepared for in vivo viral titer assessment as described above. The homogenate supernatant was used for ELISA and western blot assay. TNF-α (eBioscience, San Diego, CA) and IFN-β (PBL Assay Science, Township, NJ) ELISA was performed according to manufacturer's instructions. H2O2 measurement was performed using a hydrogen peroxide chemiluminescent kit (Enzo Life Sciences, Farmingdale, NY) following manufacturer's instructions. ELISA and H2O2 data were corrected for tumor weight by dividing concentration by total tumor weight. Western blot was performed as previously described [15 (link)]. Briefly, 4x LDS buffer (Invitrogen, Carlsbad, CA) containing β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) was added to 1x and 5% (vol/vol) concentrations, respectively and gel electrophoresis using the Novex (Invitrogen, Carlsbad, CA) western blotting system was performed. A Licor Odyssey Fc imager (Licor, Lincoln, NE) was used to image immunoblots. Antibodies used for immunoblot recognized Nitric oxide synthase (NOS), Stat1-p (Y701), Stat1 (Cell Signaling Technologies, Danvers, MA); Tubulin (Sigma-Aldrich, St. Louis, MO); and myeloperoxidase (MPO; R&D Biosystems, Minneapolis, MN).
5
Western Blot Analysis of Mouse Proteins
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Protein samples from mouse tissue were prepared by diluting 20–40 μg of protein with 1X Laemmli buffer (Biorad) supplemented with beta-mercaptoethanol (Sigma) and Ultrapure water (Gibco) to a final volume of 20–30 μL. Samples were then boiled at 95°C for 5 minutes, cooled on ice, and centrifuged briefly. Samples were then loaded into 4–20% Criterion TGX Midi protein gels (Biorad) along with Precision Plus Dual Color Protein ladder (Biorad) and run at 80–120V. Protein gels were then transferred onto iBlot2 Nitrocellulose membranes (Thermo Fisher Scientific) using an iBlot2 transfer device (Thermo Fisher Scientific). Membranes were cut to size and blocked for at least 30 minutes at room temperature in Intercept TBS Blocking buffer (LI-COR Biosciences) on a shaker. Membranes were submerged in primary antibodies diluted in blocking buffer with 0.05% Tween20 (Sigma) and incubated at 4°C overnight. Following primary antibody incubation, membranes were washed in wash buffer (1X tris buffered saline with 0.05% Tween-20). Membranes were incubated in secondary antibodies diluted in the wash buffer at room temperature for 2 hours. All primary and secondary antibody information is listed in Supplementary Tables 3 –4 . Blots were imaged using a LI-COR Odyssey Clx or Fc Imager and bands were quantified using Image Studio Lite software.
6
Immunoblotting Protocol for MOMP and ApoA1
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Western blotting and dot blotting were performed using PVDF membranes (MilliporeSigma, Burlington, MA, USA). For Western blotting, samples were resolved with SDS-PAGE. The gels were transferred using the iBlot system (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. Blots were incubated overnight at 4 °C in Odyssey Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA) containing 0.2% Tween 20 and either 0.5 mg/mL mAb40 [monoclonal antibody to the variable domain 1 (VD1) of MOMP] or 0.2 mg/mL Penta-His antibody (Qiagen, Hilden, Germany) directed against the ApoA1 His-tag, diluted 1:1000 for mAb40 and 1:500–1:1000 for Penta-His. Blots were washed 3 times for 5 min with PBS-T (50 mm NaH2PO4, 300 mm NaCl, 0.2% Tween 20, pH 7.4) and incubated for 1 h in blocking buffer containing 1 mg/mL IRDye 800CW goat (polyclonal) anti-mouse IgG (heavy and light) (LI-COR Biosciences, Lincoln, NE, USA. 1:10,000). Blots were washed again with PBS-T and imaged with a LI-COR Fc imager at 800 nm. For dot blots, 3 μg of purified MOMP-tNLP and empty tNLP were blotted using the Bio-Dot apparatus (1706545, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Blots were developed using the same methods described for Western blotting.
7
Phospho-RTK profiling in tamoxifen-resistant cells
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MCF7/TamR cells were treated with either Tamoxifen (Tam, 1 mM), TVB-3166 (200 nM), or a combination for 72 hours, then relative levels of RTK-phosphorylation were determined using the Proteome Profiler Human Phospho-RTK Array kit following the manufacturer's protocol (R&D Systems). The phospho-RTK array membranes were imaged using a Licor Fc imager with chemiluminescence. Analysis was performed using Licor's Studio Imager Lite, where background corrected mean pixel density is depicted as signal.
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