Pepscan libraries of the C-terminal region of TAF8 (residues 105–310) were immobilized on cellulose membranes via double β-alanine anchors and assembled using the SPOT technology (AG Molekulare Bibliotheken, Charité—Universitätsmedizin Berlin, Germany). Overlapping 20-mer peptides of TAF8 were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) chemistry with an offset of three amino acids between neighbouring spots. Low-density hexa-Histidine peptides were used as controls. Pepscan membranes were blocked in blocking buffer (50 mM Tris-HCl, pH 7.6, 500 mM NaCl, 20% (w/v) sucrose, 3% (w/v) bovine serum albumin) for 1 h at 4 °C, washed with TBS (50 mM Tris-HCl, pH 7.6, 500 mM NaCl) and incubated for 1.5 h with His-tagged TAF2 (10 μg ml−1) in blocking buffer or with blocking buffer alone. Membranes were incubated with mouse anti-His monoclonal primary antibody (Sigma-Aldrich, catalogue number H1029, dilution 1:3,000) and peroxidase-conjugated anti-mouse secondary antibody (Sigma-Aldrich, catalogue number A5906, dilution 1:10,000) in blocking buffer. Membranes were washed three times with TBS between each incubation step. Luminol solution (Pierce) was added and luminescence detected on a KODAK 4000MM photoimager. Images were analysed using the Dot Blot Analyzer tool in ImageJ.
Peroxidase conjugated anti mouse secondary antibody
Manufactured by Merck Group
Sourced in United States, Italy
The Peroxidase-conjugated anti-mouse secondary antibody is a laboratory reagent used to detect and quantify mouse primary antibodies in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry. This secondary antibody is conjugated with the enzyme horseradish peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of the target mouse primary antibody.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Anti ha agarose beads, by Merck Group (1 mentions)
Anti rabbit peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Anti flag agarose beads, by Merck Group (1 mentions)
Anti cullin 1, by Cell Signaling Technology (1 mentions)
Anti nfkb1, by Cell Signaling Technology (1 mentions)
Anti nfkb2, by Cell Signaling Technology (1 mentions)
Polyclonal and monoclonal anti flag antibodies, by Merck Group (1 mentions)
Anti nfatc1, by Cell Signaling Technology (1 mentions)
Anti ikkα β, by Cell Signaling Technology (1 mentions)
Anti ha monoclonal antibody, by Fortrea (1 mentions)
Peroxidase substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Tween 20, by Avantor (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
0.5 mm glass bead, by Biospec (1 mentions)
Anti survivin antibody, by Santa Cruz Biotechnology (1 mentions)
Mini trans blot transfer system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
11 protocols using peroxidase conjugated anti mouse secondary antibody
1
Mapping TAF8 Binding Sites on TAF2
2
Antibody Procurement for Cellular Signaling
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Anti-CYLD (D1A10), anti-IKKα, anti-IKKα/β, anti-NFATc1, anti-NF-kB1, anti-NF-kB2, anti-β-TRCP1 (D13F10) and anti-Cullin 1 antibodies were purchased from Cell Signaling Technology. Anti-c-Myc (9E10) and polyclonal anti-HA antibodies (Y-11) were purchased from Santa Cruz Biotechnology. Anti-Tubulin, anti-Vinculin, polyclonal and monoclonal anti-Flag antibodies, peroxidase-conjugated anti-mouse secondary antibody, peroxidase-conjugated anti-rabbit secondary antibody, anti-Flag agarose beads, and anti-HA agarose beads were purchased from Sigma-Aldrich. Monoclonal anti-HA antibody was purchased from Covance. Anti-pSer436-CYLD and pSer432/436-CYLD antibodies were developed in collaboration with Cell Signaling Technology.
3
Western Blot Analysis of GFAP
4
Western Blot Analysis of LbSemiSWEET Proteins
5
Dual Immunostaining for FAH and BrdU
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Double staining for FAH and BrdU incorporation was performed using sequential staining for FAH and BrdU incorporation on 5 µm thick slices of formalin-fixed tissue. To this end, an antibody directed against FAH (1:1.000 dilution; catalog no. 20-0034; Yecuris, Tualaton, OR, USA) was used in combination with a biotinylated anti-rabbit antibody (1:200, catalog no. 111-065-003; Dianova, Hamburg, Germany) and streptavidin-conjugated alkaline phosphatase (1:200, catalog no. 016-050-084; Dianova, Hamburg, Germany) using FastRed (Kementec, Taastrup, Denmark) as a substrate. Subsequent BrdU staining was performed as previously described using an anti-BrdU antibody (1:100; catalog no. M0744; Agilent/Dako, Santa Clara, CA, USA) together with a peroxidase-conjugated anti-mouse secondary antibody (1:100; catalog no. A2554; Sigma, Taufkirchen, Germany) and diaminobenzidine as a substrate. Slices were counterstained with hematoxylin. For analysis of BrdU incorporation, representative images of stained slices were acquired and at least 750 cells per animal were counted to determine the BrdU labeling index (i.e., the percentage of BrdU-positive nuclei).
6
Survivin Expression in Laser-Irradiated Cells
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MB49 cells were seeded onto a six-well plate and cultured until the confluence was 80% to 90% (which occurred after approximately 24 h). At that point, UCPs/siRNA-DMNPE complex was added and the cells were incubated for different time point as indicated. Then, the cells were irradiated with or without 980 nm laser and incubated at 37°C for another 24 hours. The cells were collected using cold cell lysis reagent (Pierce) as per the manufacturer's protocol. Proteins in cell lysates were separated by SDS-PAGE on 12% acrylamide gels using a discontinuous buffer system. They were then transferred to a nitrocellulose membrane (Pierce) in transfer buffer (20 mM Tris-HCl, 190 mM glycine, 20% methanol, pH 8.3) using a Mini Trans-blot transfer system (Bio-Rad) at 100 V for 1 h. The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at RT for 1 h and then incubated with anti-survivin antibody (Santa Cruz, 1∶200 dilution) at RT for 1 h. After three washes in TBST, the membranes were incubated with peroxidase-conjugated anti-mouse secondary antibody (Sigma, 1∶8000 dilution) at RT for another 1 h. Three additional washes in TBST were performed before the NC membrane were visualized with ECL solution (Pierce).
7
Western Blot Protein Quantification
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Protein separation was carried out on 50 µg samples of total protein in a 12% tris-glycine gel (Thermo Scientific, Milan, Italy). Protein was transferred onto nitrocellulose membrane (Macherrey-Nagel, Düren, Germany) overnight at 12 mV. Membranes were blocked with a buffer containing 5% fat-free milk and then incubated overnight at 4°C with a mouse anti-TP primary antibody (Thermo Scientific, Milan, Italy) at a final concentration of 2 µg/ml. Membranes were washed three times and incubated with an anti-mouse peroxidase-conjugated secondary antibody (Sigma Aldrich, Milan, Italy). Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) on a ChemiDoc MP System and quantified by Image Lab software version 4.0 (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were expressed relative to total protein and/or to the intensity of GAPDH detected on the same membrane following stripping with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific, Pittsburgh, PA, USA) and overnight incubation with GAPDH antibody at 4°C at a 1∶1000 dilution (Abcam, Cambridge, UK). Each assay was conducted in technical triplicate.
8
GPR110 Agonist Binding Assay
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HEK293 cells overexpressing human GPR110-HA WT or mutants were lysed in PBS containing 0.5% Triton X-100 and protease inhibitors. The lysate was treated with 1 µM biotinylated synaptamide (G1) or biotin at 25 °C for 30 min followed by incubation with Dynabeads M-280 streptavidin at 25 °C for 30 min. The beads were washed in the lysis buffer with mild shaking for 10 min followed by three quick washes without shaking at room temperature. The beads were incubated with 2X LDS sample buffer (Life Technologies) at 37 °C for 30 min. The G1-bound GPR110 was then detected by western blotting using anti-HA antibody (Santa Cruz Biotech., Cat.# sc-7392, 1/200 ratio) and anti-mouse peroxidase-conjugated secondary antibody (Sigma, Cat.# A4416, 1/500 ratio). Background level obtained from biotin control sample was subtracted for quantitation purpose. Statistical analysis was performed using Student’s t-test.
9
Validating Recombinant Glycoprotein Expression
10
Western Blot Analysis of Cellular and EV Markers
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Cells or EVs aliquots were lysed in RIPA buffer containing protease inhibitors (Sigma, Oakville, Canada). Equal amounts of proteins were resolved on 10% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad). Membranes were blocked in TBS containing 5% non-fat dry milk and exposed overnight at 4 °C to mouse-anti-Vimentin (ab8978, Abcam), rabbit-anti-CDH1 (3195, Cell Signaling), Rabbit anti-GM130 (ab52649, abcam), Mouse anti-TSG101 (ab83, abcam), Mouse anti-Alix (2127, Cell Signaling), or mouse-anti-β-Actin (A5316, Sigma) antibodies. Membranes were washed in TBST (TBS-0.05% Tween-20) and incubated with either anti-rabbit or anti-mouse peroxidase-conjugated secondary antibody (Sigma) for 1 h at room temperature and the blots were developed using Immobilon Western HRP Substrate (Millipore).
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