The protein samples used for western blot were extracted with RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Roche, China). Protein concentrations were determined with the bicinchoninic acid (BCA) assay kit (Thermo scientific, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinyl fluoride membranes (Merck KGaA, Darmstadt, Germany), and incubated with primary (rabbit anti-Thy-1 (ab225, Abcam, US), mouse anti- integrin β3 (sc-46655, Santa Cruz, USA), rabbit anti-LC3 I/II (L7453, Sigma, USA), rabbit anti-p62/SQSTM1 (#5114, Cell Signaling Technology, USA), rabbit anti-Akt (#4691, Cell Signaling Technology, USA), rabbit anti-p-Akt (#4060, Cell Signaling Technology, USA), rabbit anti-mTOR (#2983, Cell Signaling Technology, USA), rabbit anti-p-mTOR (#2971, Cell Signaling Technology, USA) and mouse anti-GAPDH (30201ES20, Yeasen, China) antibodies, respectively. This was followed by incubation with the appropriate secondary antibodies, including goat anti-mouse (A0216, Beyotime, China) and goat anti-rabbit (A0208, Beyotime, China) antibodies, respectively. Signals were detected using the ECL Plus Western blotting system kit (Beyotime Biotechnology, China); band intensity was measured with the Image LabTM software (Bio-Rad, USA).
Mouse anti gapdh
Manufactured by Yeasen
Sourced in United States
Mouse anti-GAPDH is a primary antibody that recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, a commonly used housekeeping protein. This antibody is used as a control in various immunoassays and Western blot analyses to detect and quantify GAPDH expression levels in samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
A0216, by Beyotime (2 mentions)
Rabbit anti p akt, by Cell Signaling Technology (2 mentions)
Rabbit anti mtor, by Cell Signaling Technology (2 mentions)
Rabbit anti lc3 1 2, by Merck Group (2 mentions)
Rabbit anti sqstm1 p62, by Cell Signaling Technology (2 mentions)
Rabbit anti p mtor, by Cell Signaling Technology (2 mentions)
A0208, by Beyotime (2 mentions)
Ab225, by Abcam (1 mentions)
Polyvinyl fluoride membrane, by Merck Group (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Vazyme (1 mentions)
Mouse anti β actin, by Yeasen (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Vazyme (1 mentions)
Cilengitide, by Selleck Chemicals (1 mentions)
3 protocols using mouse anti gapdh
1
Western Blot Analysis of Cellular Proteins
2
Comprehensive Antibody Immunostaining Protocol
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All chemicals and reagents were obtained from Sigma unless otherwise stated.
Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology). Dilution ratio for each primary antibody is in TableS1 .
Fluorescent labelled secondary antibodies were all purchased from Jackson ImmunoResearch. The stock conc. was 1.5 mg/mL and used at 1:750 dilution: Cy2‐conjugated donkey anti‐mouse IgG (Code: 715‐225‐150); Cy2‐conjugated donkey anti‐rabbit IgG (Code: 711‐225‐152); Cy5‐conjugated donkey anti‐mouse IgG (Code: I715‐175‐150); and Rhodamine (TRITC)‐conjugated donkey anti‐rabbit IgG (Code: 711‐025‐152). Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG and goat anti‐mouse IgG secondary antibodies were all purchased from Vazyme and used at 1:5000 dilution.
Primary antibodies were mouse anti‐β‐actin (Cat#: A5316‐100); mouse anti‐GAPDH (Cat#: 30201ES60; YEASEN); mouse polyclonal a anti‐α‐Tubulin (cat#:G3115); mouse polyclonal a anti‐β‐Tubulin (cat#:sc‐5274); mouse monoclonal anti‐alpha tubulin (Acetyl Lys40) (cat#:bsm‐33235M; Bioss); rabbit polyclonal anti‐mphosh6 (cat#:10695‐1‐AP; Proteintech); rabbit polyclonal anti‐BubR1 (cat#:ab28193; Abcam); human anti‐centromere CREST (Cat#: 15‐234; Antibodies Incorporated); mouse polyclonal anti‐cyclin B1 (Cat#: B8566); rabbit polyclonal anti‐Phosphorylation CDK1 (Thr14) (Cat#: OAAN02724; Aviva Systems Biology); and rabbit anti‐Phosphorylation AKT (s473) (Cat#: 9271; Cell Signaling Technology). Dilution ratio for each primary antibody is in Table
Fluorescent labelled secondary antibodies were all purchased from Jackson ImmunoResearch. The stock conc. was 1.5 mg/mL and used at 1:750 dilution: Cy2‐conjugated donkey anti‐mouse IgG (Code: 715‐225‐150); Cy2‐conjugated donkey anti‐rabbit IgG (Code: 711‐225‐152); Cy5‐conjugated donkey anti‐mouse IgG (Code: I715‐175‐150); and Rhodamine (TRITC)‐conjugated donkey anti‐rabbit IgG (Code: 711‐025‐152). Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG and goat anti‐mouse IgG secondary antibodies were all purchased from Vazyme and used at 1:5000 dilution.
3
Integrin β3 Inhibitor Regulates Autophagy Pathways
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Lipopolysaccharide (Escherichia coli O127:B8) was purchased from Sigma (USA). The integrin β3 inhibitor cilengitide (#S7077) was purchased from Selleckchem (USA). The primary antibodies used in this study were: rabbit anti-Thy-1 (ab225, Abcam, USA), mouse anti- integrin β3 (sc-46655, Santa Cruz, USA), rabbit anti-LC3 I/II (L7453, Sigma, USA), rabbit anti-p62/SQSTM1 (#5114, Cell Signaling Technology, USA), rabbit anti-Akt (#4691, Cell Signaling Technology, USA), rabbit anti-p-Akt (#4060, Cell Signaling Technology, USA), rabbit anti-mTOR (#2983, Cell Signaling Technology, USA), rabbit anti-p-mTOR (#2971, Cell Signaling Technology, USA) and mouse anti-GAPDH (30201ES20, Yeasen, China). Goat anti-mouse (A0216, Beyotime, China) and goat anti-rabbit (A0208, Beyotime, China) secondary antibodies were used as well.
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