The intact cochleae were freshly isolated after sacrificing the mice with an overdose of anesthesia and lysed with a lysis buffer containing 2% SDS, 10 mM dithiothreitol, 10% glycerol, a trace amount of Bromophenol Blue and 50 mM Tris HCl, pH 7.4 in 4°C for 1 hour. After homogenization and centrifugation, the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to PVDF membrane. The membrane was respectively probed with mouse anti-Pou4f3 (Santa Cruz Biotechnologies, CA, USA) and mouse anti-GAPDH (BioWorld, MN, USA) antibodies followed by incubation with a corresponding secondary antibody. The signals were visualized by incubation with the ECL substrate (Mucyte, China).
Mouse anti gapdh
Manufactured by Bioworld Technology
Sourced in United States, China
Mouse anti-GAPDH is a primary antibody that recognizes the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a loading control or reference protein in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti myod1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti klf4, by Bioss Antibodies (2 mentions)
Ecl western blotting substrate, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Mouse anti pou4f3, by Santa Cruz Biotechnology (1 mentions)
Hrp substrate, by Merck Group (1 mentions)
Rabbit anti tnf α, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Rabbit anti cyclin d1, by Cell Signaling Technology (1 mentions)
Mouse anti iκbα, by Cell Signaling Technology (1 mentions)
Mouse anti pcna, by Cell Signaling Technology (1 mentions)
Rabbit anti atf3, by Santa Cruz Biotechnology (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Anti visfatin, by Abcam (1 mentions)
Gw9662, by Merck Group (1 mentions)
7 protocols using mouse anti gapdh
1
Western Blot Analysis of Pou4f3 in Mice Cochleae
2
Western Blot Analysis of RASSF6 Protein
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Total protein was lysed in RIPA buffer (protease inhibitor added). A BCA assay kit (Pierce, Rockford, IL) was used to quantify protein concentration. Equal amounts of protein (20 or 30 μg) were separated on a 12% SDS–PAGE gel and then electroblotted onto nitrocellulose membranes. After blocking with 5% nonfat-dried milk in tris-buffered saline with 0.05% Tween20 (TBST) buffer for 1 h at room temperature, the membranes were incubated overnight at 4°C with rabbit anti-RASSF6 (1:200, Sigma, St Louis, MO) or mouse anti-GAPDH (1:2000, Bioworld, Beijing, China). The membranes were incubated with corresponding IgG-HRP secondary antibody (1:5000, Bioworld, Beijing, China) after 3 washes with TBST. The bands were visualized with HRP Substrate (Millipore Corporation, USA) and then exposed to KODAK film. Quantification was carried out using the imaging program Image J (NIH) and the value was then normalized to corresponding GAPDH control.
3
Western Blot Analysis of MYOD1, KLF4 and GAPDH
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Cultured cells were washed with PBS and homogenized with RIPA buffer (Beyotime, Jiangsu, China) containing protease inhibitor cocktail (Beyotime, Jiangsu, China). Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were denatured and subjected to 10% polyacrylamide gel and transferred to methanol-activated PVDF membranes. Blots were probed using the primary antibodies: mouse anti-MYOD1 (1:500; Santa Cruz Biotechnology, USA, Cat# sc-377460), rabbit anti-KLF4 (1:500; Bioss, Beijing, China, Cat# 52850R), mouse anti-GAPDH, (1:5000; Bioworld, St Louis Park, MN, USA, Cat#MB001), overnight at 4 °C. After 1 h incubation with anti-mouse or anti-rabbit HRP-conjugated second antibody (1:5000, Bioss, Beijing, China, Cat# 40296G, 40295G). Immunodetection was performed using enhanced chemiluminescence (ECL) Western blotting substrate (Beyotime, Jiangsu, China), and was detected with FluoChem R imaging system (ProteinSimple, CA, USA).
4
Western Blotting Analysis of Cellular and Tissue Proteins
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Western blotting analysis was performed as previously described.25 (link) Cellular and mouse tissue proteins were exposed to radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.6], 150 mmol/L NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS), homogenized on ice and centrifuged. The supernatants were resolved via 10% SDS‐PAGE and were transferred to PVDF membranes (Millipore). The membranes were blocked with Tris‐buffered saline (TBS) containing 5% non‐fat milk and were incubated with the following primary antibodies: rabbit anti‐IRF7 (sc‐9083; 1:200; Santa Cruz), mouse anti‐PCNA (#2586; 1:1000; Cell Signaling Technology), rabbit anti‐Cyclin D1 (#2978; 1:1000; Cell Signaling Technology), rabbit anti‐NF‐κB p65 (#4764; 1:1000; Cell Signaling Technology), rabbit anti‐phosphorylated‐NF‐κB p65 (ser536) (BS4138; 1:1000; Bioworld), mouse anti‐IκBα (#4814; 1:1000; Cell Signaling Technology), mouse anti‐phosphorylated IκBα (BS4105; 1:1000; Bioword), rabbit anti‐TNFα (#3707; 1:1000; Cell Signaling Technology), goat anti‐IL‐6 (AF‐406‐NA; 1:500; R&D System), goat anti‐Collagen type I (sc‐8784; 1:1000; Santa Cruz), goat anti‐Collagen type III (sc‐8781; 1:1000; Santa Cruz), rabbit anti‐ATF3 (sc‐188; 1:200; Santa Cruz), and mouse anti‐GAPDH (MB001; 1:10 000; Bioworld).
5
Protein Expression Analysis in Chicken Cells
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Cultured cells were washed with PBS and homogenized with RIPA buffer (Beyotime, Jiangsu, China) containing protease inhibitor cocktail (Beyotime, Jiangsu, China). Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, Jiangsu, China). Proteins were denatured and subjected to 10% polyacrylamide gel and transferred to methanol-activated PVDF membranes. Blots were probed using the primary antibodies: mouse anti-MYOD1 (1:500; Santa Cruz Biotechnology, USA, Cat# sc-377460), rabbit anti-KLF4 (1:500; Bioss, Beijing, China, Cat# 52850R), mouse anti-GAPDH, (1:5000; Bioworld, St Louis Park, MN, USA, Cat#MB001), overnight at 4 °C. After one h incubation with anti-mouse or anti-rabbit HRP-conjugated second antibody (1:5000, Bioss, Beijing, China, Cat# 40296G, 40295G). Immunodetection was performed using enhanced chemiluminescence (ECL) Western blotting substrate (Beyotime, Jiangsu, China) and detected with FluoChem R imaging system (ProteinSimple, CA, USA). Trimmed reads were mapped to the chicken reference genome (Ensembl release 100:ftp://ftp.ensembl.org/pub/release100/fasta/gallus_gallus/dna/Gallus_gallus.GRCg6a.dna.toplevel.fa.gz) using HISAT2 [27] . Read counts for each gene were calculated using Stringtie (v.2.1.2) and normalized by library sequencing depth using the R package DESeq2 (v.1.28.1) after ltering the gene with low expression
6
FPR2 Receptor Signaling Pathway
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Recombinant human apo-SAA1 was purchased from PeproTech (Rocky Hill, NJ) and Sigma-Aldrich (St. Louis, MO). Rabbit monoclonal anti-Visfatin was purchased from Abcam (Cambridge, UK). Mouse polyclonal antibody for peroxisome proliferator-activated receptor-γ (PPAR-γ) was purchased from Bioss (Beijing, China). Visfatin Elisa Kit was also purchased from Bioss (Beijing, China). Rabbit polyclonal antibodies for total-ERK1/2 and p-ERK1/2 were both purchased from Cell Signaling Technology Inc. (Danvers, MA). Mouse anti-GAPDH was purchased from Bioworld Technology (Minneapolis, USA). FPR2 siRNA for both mice and humans was purchased from Santa Cruz Biotechnology (Dallas, TX). H2N-WRWWWW-CONH2 (WRW4), a FPR2 antagonist, was purchased from Tocris Bioscience (Ellisville, Missouri). WKYMVm trifluoroacetate salt, a peptide agonist of formyl peptide receptors, was purchased from Sigma-Aldrich (St. Louis, MO). PD98059 (a selective ERK1/2 inhibitor) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). PPAR-γ agonist Rosiglitazone and PPAR-γ antagonist GW9662 were purchased from Sigma-Aldrich (St. Louis, MO).
7
Western Blot Analysis of Cell Proteins
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Cultured cells were collected and lysed in lysis buffer containing a proteinase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF, Cell Signaling Technology, Danvers, MA, USA). Equal amounts of proteins were separated on 8–12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and then were transferred to PVDF membranes (Millipore, Boston, MA). The membranes were then blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After that, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and were visualized with enhanced chemiluminescence as previously described.36 (link) The antibodies used in this study were as follows: rabbit anti-integrin alpha 2 (ab133557, Abcam), rabbit anti-integrin beta 1 (#9699, Cell Signaling Technology), mouse anti-GAPDH (MB001, Bioworld Technology), rabbit anti-HSP70 (BS6446, Bioworld Technology), rabbit anti-CD9 (#13174, Cell Signaling Technology), mouse anti-CD63 (ab193349, Abcam), rabbit ant-Tsg 101 mAb (E303, Bioworld Technology), HRP-conjugated goat anti-rabbit antibody (#7074, Cell Signaling Technology), and HRP-conjugated goat anti-mouse antibody (BS12478, Bioworld Technology).
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