Samples were lysed with EZ lysis buffer (1 M Tris at pH 7, 50% glycerol, 20% SDS, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined using the protein assay kit (Bio-Rad, 500-0006). Equal amounts of proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, 10401197). Membranes were incubated overnight at 4°C with primary antibodies and for 1 h at room temperature with secondary HRP-conjugated antibodies. The antibodies used in this study include IGF1R, BCL-2, and CDC25A from Santa Cruz Biotechnology (sc-492, sc-713, and sc-7389, respectively); p-Akt Ser473, p-IGF1R Tyr1131, and p-IGF1R Tyr1135 from Cell Signaling (4060, 3021, and 3918, respectively); and β-Actin from US Biological (A0760-40).
β actin
Manufactured by US Biological
Sourced in China, United States
β-actin is a highly conserved cytoskeletal protein that is ubiquitously expressed in all eukaryotic cells. It is a key component of the microfilament system and plays a crucial role in various cellular processes, such as cell motility, structure, and division.
Automatically generated - may contain errors
Lab products found in correlation
P akt ser473, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Igf1r, by Santa Cruz Biotechnology (1 mentions)
P igf1r tyr1135, by Cell Signaling Technology (1 mentions)
Cdc25a, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab2413, by Abcam (1 mentions)
Ab5831, by Abcam (1 mentions)
Ab6528, by Abcam (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ab66043, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Horseradish peroxidase conjugated igg, by Bio-Rad (1 mentions)
Ab54230, by Abcam (1 mentions)
Ab133497, by Abcam (1 mentions)
Sc 1458, by Santa Cruz Biotechnology (1 mentions)
A0760 40, by US Biological (1 mentions)
4 protocols using β actin
1
Western Blot Analysis of Cell Signaling
2
Protein Expression Analysis via Western Blot
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Cells were homogenized in 1X radioimmunoprecipitation assay buffer and western blotting was performed to analyze the protein expression. Briefly, protein concentrations were examined using a BCA protein assay (Invitrogen; Thermo Fisher Scientific, Inc.) and protein samples (40 µg) was separated by 15% SDS-PAGE. Proteins were transferred into nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the primary antibodies used in the immunoblotting assays were TGF-β1 (1:1,000, ab66043), ERK (1:2,000, ab54230), AKT (1:2,000, ab8805), cadherin (1:1,000, ab6528), fibronectin (1:1,000, ab2413), α-smooth muscle actin (α-SMA; 1:1,000, ab5831; all from Abcam, Shanghai, China) and β-actin (1:2,000, G8140; United States Biological, Salem, MA, USA) for 12 h at 4°C following blocking with 5% skimmed milk for 1 h at 37°C. Horseradish peroxidase-conjugated IgG (Bio-Rad Laboratories, Inc.) was used at a 1:5,000 dilution for 2 h at 37°C and detected using a Western Blotting Luminol reagent (sc-2048; Santa Cruz Biotechnology Inc., Dallas, TX, USA).
3
Protein Expression Quantification Protocol
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A previously described procedure was used24 with the following primary antibody (SERT: 1:1000, sc‐1458, Santa Cruz Biotechnology; Flotillin‐1:1:1000, ab133497, Abcam; β‐actin: 1:2000, A0760‐40, US Biological, Salem, Massachusetts) which were incubated with membranes overnight at 4°C. Target protein densitometry values were normalized to those of the housekeeping protein to semi‐quantitatively determine protein levels.
4
Western Blot Analysis of SERT Protein
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Immediately after the last PET scan, brains were rapidly extracted and the hippocampus, cerebellum, and cortex were dissected. Tissue samples were then snap-frozen in liquid nitrogen and stored at −80 °C until used for western blot analysis.
Total protein was extracted from powderized brain tissue and 50 µg of protein were loaded and subjected to SDS-polyacrylamide gel electrophoresis according to a previously described procedure34 (link). Membranes were incubated overnight with the primary antibodies (SERT, target: sc-1458, dilution 1:500; secondary antibody: anti-goat IgG-HRP, sc-2020, dilution 1:2000; both Santa Cruz Biotechnology, Inc, Dallas, TX, USA; β-actin, housekeeping protein: A0760-40, dilution 1:2000; US Biological, Swampscott, MA, USA; secondary antibody: anti-mouse IgG-HRP, #7076, dilution 1:5000; Cell Signaling Technology, Danvers, MA, USA) used as the housekeeping protein. Chemiluminescent imaging was performed using a FluorChem HD2 imager (Alpha Innotec, Kasendorf, Germany) and densitometry values were determined using the software ImageJ (NIH, Bethesda, MD, USA). SERT protein densitometry values for each sample were normalized to the corresponding housekeeping protein value to obtain semiquantitative measures of SERT expression.
Total protein was extracted from powderized brain tissue and 50 µg of protein were loaded and subjected to SDS-polyacrylamide gel electrophoresis according to a previously described procedure34 (link). Membranes were incubated overnight with the primary antibodies (SERT, target: sc-1458, dilution 1:500; secondary antibody: anti-goat IgG-HRP, sc-2020, dilution 1:2000; both Santa Cruz Biotechnology, Inc, Dallas, TX, USA; β-actin, housekeeping protein: A0760-40, dilution 1:2000; US Biological, Swampscott, MA, USA; secondary antibody: anti-mouse IgG-HRP, #7076, dilution 1:5000; Cell Signaling Technology, Danvers, MA, USA) used as the housekeeping protein. Chemiluminescent imaging was performed using a FluorChem HD2 imager (Alpha Innotec, Kasendorf, Germany) and densitometry values were determined using the software ImageJ (NIH, Bethesda, MD, USA). SERT protein densitometry values for each sample were normalized to the corresponding housekeeping protein value to obtain semiquantitative measures of SERT expression.
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