Total proteins were collected from tissues and cultured cells using RIPA buffer (Solarbio, Beijing, China). The concentrations of proteins were measured using bicinchoninic acid (BCA) assay kit (Beyotime, Biotechnology, Shanghai, China). The proteins were separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and electro-transferred on to polyvinylidene fluoride (PVDF) membrane (Reno, Hangzhou, China) in a semi-dry blotting apparatus (Bio-Rad, Hercules, California). After blocking with TBST-soluble 5% dried skimmed milk at room temperature for 2 h, the membranes were respectively bound to the mouse monoclonal anti-human SOX12 antibody (1:1000; Abcam, Cambridge, MA, U.S.A.) or mouse monoclonal anti-human GAPDH (1:5000; Santa Cruz Biotechnology Inc, CA, U.S.A.) at 4°C overnight. Then membrane was bound to the corresponding secondary antibodies (Rabbit anti-mouse IgG, 1:6000; Abcam) at 37°C for 1 h. The blots were assessed using an enhanced chemiluminescence reagent (ECL, GE Healthcare, Chicaogo, IL, U.S.A.).
Mouse monoclonal anti human gapdh
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse monoclonal anti-human GAPDH is a primary antibody that specifically recognizes the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a loading control or housekeeping gene in various biological assays.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor, by Merck Group (2 mentions)
Bca protein assay kit, by Vigorous Biotechnology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Mouse monoclonal anti human irs 1, by Santa Cruz Biotechnology (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Rabbit anti mouse igg, by Abcam (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Semi dry blotting apparatus, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti mouse immunoglobulin g igg, by Santa Cruz Biotechnology (1 mentions)
Spss software version 19, by IBM (1 mentions)
5 protocols using mouse monoclonal anti human gapdh
1
Protein Expression Analysis by Western Blot
2
Protein Isolation and Western Blot Analysis
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The total protein isolation and Western blot analyses were performed as previously described.17 (link) The membranes were probed with primary antibodies: mouse monoclonal anti-human CDK6 (1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse monoclonal anti-human GAPDH (1:5000; Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were incubated with secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000; Santa Cruz Biotechnology, Inc.). GAPDH was used as an internal control. The protein bands were observed using enhanced chemiluminescence (ECL) reagents (Super Signal Dura kit, Pierce, IL, USA) and were quantified using Image Lab™ Software (Bio-Rad).
3
Western Blot Analysis of Protein Expression
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Protein was extracted from tissues and cells using RIPA lysis buffer containing proteinase inhibitor (Sigma‐Aldrich, St Louis, MO, USA). Protein concentration was determined using the BCA Protein Assay Kit (Vigorous Biotechnology Beijing, Beijing, China). Equal amounts of protein lysates (20 μg each lane) were resolved using 10% SDS‐PAGE gels, and then electroblotted onto nitrocellulose membranes (Millipore, Madison, WI, USA). The membranes were blocked for 2 h with 5% non‐fat dry milk in Tris‐buffered saline containing 0.1% Tween‐20, and incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti‐human IRS‐1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti‐human RGS‐17 (1:500; Santa Cruz Biotechnology), and mouse monoclonal anti‐human GAPDH (1:5000; Santa Cruz Biotechnology). GAPDH was used as an internal control for protein loading. The membrane was further incubated with HRP‐conjugated goat anti‐mouse IgG (1:5000; Santa Cruz Biotechnology) for 1 h at room temperature. Immune complexes were detected by ECL (Cell Signaling Technology, Danvers, MA, USA). Integrated density of the band was quantified by Quantity One software (Bio‐Rad).
4
Western Blot Analysis of Protein Expression
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Protein was extracted from tissues and cells using RIPA lysis buffer containing proteinase inhibitor (Sigma-Aldrich, Otsu, Japan). The protein concentration was determined using the BCA Protein Assay Kit (Vigorous Biotechnology Beijing, Beijing, China). Equal amounts of protein lysates (20 μg each lane) were resolved using 10% SDS-PAGE gels and then electroblotted onto nitrocellulose membranes (Millipore, Madison, WI, USA). The membranes were blocked for 2 hr with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20, and incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti-human IRS-1 (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-human URI (1:500, Santa Cruz Biotechnology), and mouse monoclonal anti-human GAPDH (1:5,000, Santa Cruz Biotechnology). GAPDH was used as an internal control for protein loading. The membrane was further incubated with horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (1:5,000, Santa Cruz Biotechnology) for 1 hr at room temperature. The immune complexes were detected by enhanced chemiluminescence (ECL; Cell Signaling Technology, Danvers, MA, USA). The integrated density of the band was quantified by Quantity One software (Bio-Rad, Hercules, CA, USA).
5
Quantification of SOX12 and Twist Proteins
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4˚C as follows: Mouse monoclonal antihuman SOX12 (1:1,000; cat. no. Ab54371; Abcam, Cambridge, MA, USA), mouse monoclonal antihuman Twist (1:1,000; cat. no. sc-81417; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse monoclonal antihuman GAPDH (1:5,000; cat no sc-365062; Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were incubated with polyclonal goat antimouse horseradish peroxidaseconjugated immunogloblin G (1:10,000; cat. no sc-2005; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. GAPDH was used as an internal control. Protein bands were observed using an enhanced chemiluminescence reagent (ECL; GE Healthcare, Chicaogo, IL, USA). Gray analysis was performed using software Gel-Pro Analyzer 4 (United States Biochemical, Cleveland, OH, USA).
Statistical analysis. All statistical analyses were performed using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). All data are presented as the mean ± standard deviation from at least three independent experiments with similar results. Continuous data were compared using the Student's two-tailed t-test or one-way analysis of variance with post hocTukey's tests. Correlations between miR-744 expression and SOX12 expression were evaluated by Pearson's correlation analysis. In all cases, P<0.05 was considered to indicate a statistically significant difference.
Statistical analysis. All statistical analyses were performed using SPSS software, version 19.0 (IBM SPSS, Armonk, NY, USA). All data are presented as the mean ± standard deviation from at least three independent experiments with similar results. Continuous data were compared using the Student's two-tailed t-test or one-way analysis of variance with post hocTukey's tests. Correlations between miR-744 expression and SOX12 expression were evaluated by Pearson's correlation analysis. In all cases, P<0.05 was considered to indicate a statistically significant difference.
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