Cochleae were isolated from P4 Lgr5DTR-EGFP/+; GLASTCreERT/+; Rpl22HA/+ and GLASTCreERT/+; Rpl22HA/+ mice (spiral ganglia, lateral wall, and Reissner’s membrane were removed) in cold Hanks’ Balanced Salt Solution (HBSS) and then immediately flash-frozen using liquid nitrogen prior to RNA isolation. About 2 to 8 cochleae were pooled and processed for ribosome IP followed by RNA extraction as described [40 (link)]. Briefly, cochlear ducts were homogenized and incubated with 5 μg purified anti-HA.11 (BioLegend) for 6 hours at 4°C before the addition of the equivalent of 300 μl Dynabeads protein G (ThermoFisher Scientific) and further incubation overnight. Additionally, 5% of the homogenate was kept before addition of the antibody to be used as input. RNA was extracted from the IP and input samples using the RNeasy Plus Micro kit (Qiagen) following the manufacturer’s instructions. RNA quality was confirmed with a BioAnalyzer 2100 picochip (Agilent Technologies) performed at the Genomics Core Facility in the Center for Innovative Biomedical Resources (University of Maryland School of Medicine). All RNA integrity numbers (RINs) were above 8.
Anti ha 11
Manufactured by BioLegend
Sourced in United States
Anti-HA.11 is a mouse monoclonal antibody that recognizes the Influenza Hemagglutinin (HA) epitope. The HA epitope is commonly used as a protein tag for the expression and detection of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy plus micro kit, by Qiagen (4 mentions)
Bioanalyzer 2100 rna pico chip, by Agilent Technologies (3 mentions)
Anti flag m2, by Merck Group (3 mentions)
Anti lamp2, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Immunoway (2 mentions)
Anti gst, by Yeasen (2 mentions)
Anti β actin, by Yeasen (2 mentions)
Anti vegf, by Novus Biologicals (2 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (2 mentions)
Anti cd31, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer 2100 pico chip, by Agilent Technologies (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
P9599, by Merck Group (1 mentions)
Ecl plus western detection kit, by GE Healthcare (1 mentions)
Image quant las 4000 mini ccd camera system, by GE Healthcare (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Merck Group (1 mentions)
Las 4000, by Cytiva (1 mentions)
Ecl chemiluminescence kit, by PerkinElmer (1 mentions)
16 protocols using anti ha 11
1
Ribosome Immunoprecipitation from Mouse Cochlea
2
Protein Extraction and Western Blot
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Proteins were extracted in 50 mM Na-phosphate (pH 7.4), 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50 µM MG132, 2 mM Na3VO4, 2 mM NaF, and 1% (v/v) protease inhibitor mixture for plant extracts (Sigma-Aldrich, P9599). For immunoblot analysis, total cellular proteins were separated by electrophoresis in 10% (w/v) SDS–polyacrylamide gels and transferred to PVDF membranes according to the manufacturer's instructions (iBlot dry blotting system, Thermo Fisher Scientific).
Rabbit polyclonal antibodies were generated against synthetic peptides derived from the RUP2 protein sequence [amino acids 1–15 + C: MNTLHPHKQQQEQAQC; anti-RUP2(1–15)] and were affinity-purified against the peptide (Eurogentec). Anti-RUP2(1–15), anti-UVR8(426–440) (Favory et al. 2009 (link)), anti-HA.11 (BioLegend, 901513), and anti-actin (Sigma-Aldrich, A0480) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulins (Dako A/S) were used as the secondary antibodies. Chemiluminescent signals were generated with the ECL Plus Western detection kit and revealed with an ImageQuant LAS 4000 mini-CCD camera system (GE Healthcare).
Rabbit polyclonal antibodies were generated against synthetic peptides derived from the RUP2 protein sequence [amino acids 1–15 + C: MNTLHPHKQQQEQAQC; anti-RUP2(1–15)] and were affinity-purified against the peptide (Eurogentec). Anti-RUP2(1–15), anti-UVR8(426–440) (Favory et al. 2009 (link)), anti-HA.11 (BioLegend, 901513), and anti-actin (Sigma-Aldrich, A0480) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulins (Dako A/S) were used as the secondary antibodies. Chemiluminescent signals were generated with the ECL Plus Western detection kit and revealed with an ImageQuant LAS 4000 mini-CCD camera system (GE Healthcare).
3
Western Blot Analysis of p63 Protein
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Cell and tissues extracts were resolved on SDS polyacrylamide gels and blotted onto Hybond P, PVDF membrane (GE Healtcare Chicago, IL, USA). Membranes were blocked with PBST 5% non-fat dry milk, incubated with primary antibodies for 2 h at room temperature, washed and hybridized with peroxidase conjugated secondary antibodies for 1 h at room temperature (goat anti-rabbit or goat anti-mouse, Biorad Hercules, CA, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, MA, USA) The antibodies used were: anti-p63α (D2K8X, Cell Signaling Technologies, Danvers, MA, USA; 1/500 dilution), anti-p63 (D9L7L, Cell Signaling Technologies, Danvers, MA, USA; 1/250), anti-GAPDH (clone 6C5, Merck Millipore, Darmstadt, Germany), anti-HA.11 (Biolegend, San Diego, CA, USA; 1/500), and anti-βactin (AC-15, Sigma, St. Louis, MO, USA; 1/5000).
Uncropped blots for westerns and other blots can be found in the Source data file.
Uncropped blots for westerns and other blots can be found in the Source data file.
4
Single-Cell Ribosome Profiling of Noise-Exposed Cochlea
5
Antibody validation for Western blot and co-IP
6
Antibody Validation for Western Blotting
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anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-myc (Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-α-tubulin (Yeasen, 30304ES60), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-VEGF (Novus Biologicals, NB100-664), anti-PCNA (Servicebrio, GB11010), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-GFP (Yeasen, 31002ES60), anti-LUB9 (Lifesensors, AB130), anti-HIF1α (Novus, NB100-479; Abcam, ab228649), anti-HIF1β (Cell Signaling technology, #5537), anti-Otulin (Abcam, ab211328), anti-HOIP (Abcam, ab46322; R&D systems, MAB8039), anti-HOIL-1L (Millipore, MABC576), anti-Sharpin (Proteintech, 14626-1-AP), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-Lamin B1 (Zenbio, R24825), normal mouse immunoglobulin (IgG) 1 (Santa Cruz Biotechnology, sc-3877), anti-mouse IgG(H + L) (Jackson ImmunoResearch, 151383), and anti-rabbit IgG(H + L) (Jackson ImmunoResearch, 145472).
7
ELISA-based Quantification of HA-tagged hBGT1
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ELISA was performed exactly as previously described using tsA201 cells transiently expressing HA-tagged or untagged hBGT1 constructs65 (link). To this end, washed, fixated, blocked cells were incubated for 45 min with anti-HA (anti-HA.11, BioLegend, San Diego, CA, USA) diluted 1:1,000 in blocking solution, washed carefully and incubated for 45 min with a horseradish peroxidase-conjugated antibody (anti-mouse, P0447, Dako, Glostrup, Denmark) diluted 1:1,500 in blocking solution. Finally, quantification of the signal from well-washed cells were obtained using SuperSignal ELISA Femto substrate (ThermoFisher Scientific, Waltham, MA USA) by chemiluminescence in an EnSpire2300 Multilabel Reader (PerkinElmer). HA-tagged hBGT1 was used as a positive control, while untagged wt hBGT1 was used as a negative control. All experiments were performed in sextuplicate measurements in three independent experiments.
8
Western Blot Analysis of Drosophila Proteins
9
Immunofluorescence Imaging of Transfected Cells
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Transfected cells on glass coverslips were washed twice with PBS and fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 in PBS, and blocked in 2% Bovine Serum Albumin in PBS for 10 min each at room temperature. Primary antibodies anti-HA.11 (BioLegend, 901514, San Diego, CA, USA) or anti-A3H were used at a dilution at 1:500 and 1:50, respectively, in 0.1% Triton X-100 in PBS and incubated with cells for 20 min at room temperature. Cells were washed five times with 0.1% Triton X-100 in PBS prior to incubation with Alexa Fluor 488-labeled anti-mouse secondary antibody (Thermo Fisher, A-11001) at a 1:1000 dilution in 0.1% Triton X-100 in PBS. Cells were again washed five times prior to mounting glass coverslips in ProLong Gold antifade reagent containing DAPI (Thermo Fisher, P36935). Images were obtained using a Nikon E800 microscope at 40× magnification.
10
Ribosome Immunoprecipitation from Mouse Liver
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Three 30-day old Gfi1Cre/+;RiboTagHA/HA mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Livers were harvested and immediately frozen on dry ice. Equal amounts of liver were used for input RNA extractions (RNeasy Plus Micro kit, QIAGEN USA, Germantown, MD, USA) or futher processed for ribosome immunoprecipitation (5 μg of purified anti-HA.11, BioLegend, San Diego, CA, USA) followed by RNA extraction as previously decribed in Sanz et al., 2009 [16 (link)]. The RNeasy Plus Micro kit is optimized for the removal of genomic DNA through a combination of high salt buffer and the gDNA Eliminator spin column. Quality of the RNA was assessed on an Agilent Technologies Bioanalyzer 2100 RNA pico chip as per the manufacturer’s instructions (Agilent Technologies, Palo Alto, CA, USA). All samples had a RIN score of 10 and no evidence of DNA contamination in the form of a high molecular weight DNA band. All RNA was equally aliquoted to test for the performance of five commercial kits and seven protocols.
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