For immunoprecipitation experiments, WTT-CHO cells were transiently transfected with pcDNA3.1 (+) empty vector (control) or vectors encoding HA-GPCRs in similar conditions to those used for AFM-SMFS experiments. Forty-eight hours post-transfection, cells were lysed in a lysis buffer (140 mM NaCl/2 mM EDTA/25 mM Tris, pH 7.4/0.5% DDM) supplemented with complete protease and phosphatase inhibitors (Roche). Protein extracts (~2 mg) were immunoprecipitated overnight at 4 °C with DynabeadsTM Protein G (Invitrogen) precoated with an anti-HA antibody (BioLegend, 16B12 Clone) and proceeded to western-blot analysis as previously described33 (link). Proteins were detected with the anti-HA primary antibodies (Biolegend, 16B12 Clone) followed by antimouse Horse Radish Peroxidase (HRP)-conjugated secondary antibodies (TrueBlot, eB144Clone, Rockland) using enhanced chemiluminescence detection reagent (RPN2232 Prime, GE Healthcare).
Trueblot
Manufactured by Rockland Immunochemicals
TrueBlot is a proprietary Western blot detection reagent developed by Rockland Immunochemicals. It is designed to specifically detect target proteins in Western blot analysis while minimizing background signal.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeadstm protein g, by Thermo Fisher Scientific (1 mentions)
Clone 16b12, by BioLegend (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Horseradish, by GE Healthcare (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Fusion fx7 system, by Vilber (1 mentions)
Hybond c extra, by Cytiva (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Ecl plus reagent, by PerkinElmer (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Las 3000 mini, by Fujifilm (1 mentions)
Dynabeads protein g beads, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by Merck Group (1 mentions)
Anti ha affinity matrix 3f10, by Roche (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Roche (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
8 protocols using trueblot
1
GPCR Immunoprecipitation and Western Blot
2
Validation of AKI Biomarkers by Western Blot
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A different cohort to that used in the discovery phase was recruited for confirmation of candidate markers by WB. 10 mL urine samples were collected from 23 AKI patients and 13 healthy individuals. The same amount of total protein (10 μg) total protein was dissolved in Laemmli buffer and loaded per lane in a 12% acrylamide gel (4% stacking). Membranes were blocked with PBS containing 7.5% non-fat dry milk powder and 0.1% Tween-20 for 1 h at room temperature. Membranes were then incubated for 1 h with specific primary antibody rabbit monoclonal anti-KNG1 (1:5000) (Abcam) or with rabbit monoclonal anti-RBP4 (1:50000) (Abcam) in phosphate buffered saline in Tween-20 (PBS-T) (0.05%) containing 5% non-fat dry milk. Finally, they were incubated with horseradish peroxidase (HRP)-conjugated rabbit TrueBlot® (1:1000) (Rockland) for KNG1 or HRP-conjugated goat anti-rabbit (1:10000) (Nordic) for RBP4 as secondary antibody, in PBS-T (0.05%) containing 5% non-fat dry milk. Detection was performed by enhanced chemiluminescence (ECL kit; GE Healthcare) following the instructions of the manufacturer. Protein bands were quantified by ImageJ software.
Concentration of RBP4 was additionally measured by ELISA in urine from a cohort of 8 control subjects and 8 AKI patients following manufacturer’s instructions (Abcam, ab108897).
Concentration of RBP4 was additionally measured by ELISA in urine from a cohort of 8 control subjects and 8 AKI patients following manufacturer’s instructions (Abcam, ab108897).
3
DUSP10 Protein Stability Measurement
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Immunoprecipitations and Western analyses were performed as previously described [17 (link)], except that TrueBlot™ reagents (Rockland) were used for DUSP10 immunoprecipitations to avoid IgG heavy chain masking. For DUSP10 half-life determinations, cells were cultured with 35S-methionine for 45 min followed by the addition of unlabeled methionine (1 μM). DUSP10 was immunoprecipitated at the indicated time points and imaged via autoradiography. DUSP10 bands were excised and counted in a scintillation counter for quantification. Data shown are the means from three independent experiments.
4
Western Blot Protein Detection Protocol
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Protein extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membrane (Hybond-C Extra, Amersham or BioTraceNT, PALL), immunoblotted with the indicated concentration of primary antibodies and revealed using the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE healthcare) or True Blot (Rockland, Tebu-Bio). Proteins were then identified using Immobilon Western (Millipore) or Western Lightning Ultra (Perkin Helmer) chemiluminescent solutions and images acquired on a Fusion FX7 system (Vilber Lourmat). Full-size blots for cropped gels can be found in Supplementary figures 6 , 7 .
5
Protein Extraction and Western Blot Analysis
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Each tissue was dissected, weighed and homogenized by sonication in 3 ml g−1 of buffer A (10 mM Tris-HCl at pH 7.6, 0.15 M NaCl, 1% Triton X-100, protease inhibitors). The supernatant after centrifugation at 20,400g at 4 °C for 0.5 h was labelled as soluble fraction. The pellet was dissolved with sonication in 1 ml g−1 buffer B (3% SDS, 5% 2-mercaptoethanol), which was termed pellet fraction. The samples were resolved by 10%PAGE (SDS-PAGE), transferred to reinforced nitrocellulose or PVDF membranes and subjected to IB analysis using a blocking buffer containing 5% skim milk in TBST (0.1 M Tris-HCl at pH 7.4, 0.15 M NaCl, 0.05% Tween 20). Chemiluminescence detection and densitometry were performed with horseradish peroxidase-conjugated secondary antibodies, ECL-Plus reagent (PerkinElmer), TrueBlot (Rockland) and an image analyser LAS-3000mini with MultiGauge software (Fuji). Uncropped version of each image is shown in Supplementary Fig. 15 .
6
IP Assay for Protein Interactions
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Immunoprecipitation (IP) assay was carried out, according to the reported method14 (link),15 (link). HEK293T cells were transfected with each plasmid by Lipofectamine 2000. To express ORF1-EGFP, flag-tagged SIRT6 flag-tagged ovalbumin (OVA), and ST (streptavidin-tagged)-EGFP, we constructed pORF1-EGPF, pFlag-SIRT6, pFlag-OVA, and pST-EGFP. Cells were suspended in IP buffer composed of 20 mM Tris–HCl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 10% glycerol. 500 μg of cell extract were reacted with 4 μg of αFlag or EGFP or αPPARα and then recovered with Dynabeads protein G beads (Thermo scientific). About one-tenth of each extract subjected to IP was loaded onto SDS-PAGE and simultaneously analyzed as an input sample. The immunohistochemical analysis was done by the reported method14 (link),15 (link). We used the TrueBlot (Rockland Immunochemicals, Inc.) secondary antibody, which masks the detection of the heavy and light IgG chains, and thus, it displayed them as thin or undetectable bands.
7
Lysate-based Immunoprecipitation Protocol
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Cells were lysed using TNTE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1x Complete protease inhibitor (Roche), 1x PhosSTOP (Roche)) supplemented with 10% (v/v) glycerol and 0.1% (w/v) BSA and the clarified lysates used for immunoprecipitation using the indicated antibodies for 2 hr at 4°C. Antibodies were coupled to protein G Sepharose (Sigma). Pelleted beads were washed 3 times with TNTE buffer and eluted with 2x Laemmli sample buffer at 100°C for 10 min before resolving by SDS-PAGE (4%–12% Bis-Tris NuPAGE gels, Life Technologies) and western blotting. GFP-tagged proteins were immunoprecipitated using GFP-TRAP beads and HA-tagged proteins with anti-HA affinity matrix 3F10 (Roche), using the same buffer and protocol. During western blotting of IP experiments, TrueBlot (Rockland) was used to reduce background from IgG when required.
8
Immunoprecipitation of Developmental Regulators
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For each IP, 40 de-jellied embryos were collected at the developmental stage of interest. They were homogenised in 400 µl ice-cold IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA and 0.25 % Igepal CA-630) supplemented with an EDTA-free protease inhibitor cocktail (Roche, 11873580001). The extract was kept on ice for 5 min. Subsequently, the extract was mixed with 400 µl 1,1,2-trichloro-1,2,2-trifluoroethane (Sigma) by vigorously inverting the tube and cleared by centrifugation at 16,000 × g (4 °C) for 5 min. An aliquot of 20 µl supernatant was saved as input. The remaining supernatant was incubated with 0.5 µl antibody (anti-Sox361 (link), anti-VegT16 (link) or anti-Tbx6; all rabbit polyclonal antibodies) at 4 °C for 1 h. Subsequently, each embryonic extract was supplemented with 20 µl washed magnetic Dynabeads M280 conjugated to sheep anti-rabbit IgG (Thermo Fisher Scientific, 11205D) and kept on a tube rotator at 4 °C for another 2 h. Next, the beads were washed three times with IP buffer. The immunoprecipitate was eluted off the beads with 20 µl 1× SDS loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 6% glycerol and 5% β-mercaptoethanol). Western blotting was carried out with TrueBlot (Rockland Immunochemicals) and normal horseradish peroxidase (HRP)-conjugated secondary antibodies for IP and input samples, respectively.
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