Western Blot Analysis of Signaling Proteins

The western blot analysis was carried out as described by Park et al. [18 (link)]. The protein sample (20 μg) was loaded and separated by SDS–PAGE. After electrophoresis, the protein was electrotransferred to a poly vinylidene difluoride membrane (Immobilon-P; Millipore Corp., Billeria, MA). The antibodies phosphorylated Src (Y416, 1:500), total Src (1:1000), p-Rb (S780, 1:500 dilution), total Rb (1:1,000 dilution), p-IkBα (S32/36, 1:500 dilution), total IkBα (1:1,000 dilution), caspase 8 (1:1,000 dilution), and GAPDH (1:1,000 dilution) were purchased from Cell Signaling Technology (Denver, MA). Antibodies for VEGF (1:200 dilution) and TNFα (1:200 dilution) were purchased from Santa Cruz Biotechnology. Antibody for cyclin D1 (1:500 dilution) and TRβ (1:500) were purchased from Neomarkers (Thermo Scientific, Cambridge, MA) and Rockland, respectively. The blots were stripped with Re-Blot Plus (Millpore, Billeria, MA) and reprobed with rabbit polyclonal antibodies to GAPDH. Band intensities were quantified by using NIH IMAGE software (Image J 1.47).

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Park J.W., Zhao L., Willingham M.C, & Cheng S.Y. (2016). Loss of Tyrosine Phosphorylation at Y406 Abrogates the Tumor Suppressor Functions of the Thyroid Hormone Receptor β. Molecular carcinogenesis, 56(2), 489-498.

Publication 2016

Immobilon-P total Src total Rb total IkBα caspase 8 GAPDH Antibodies for VEGF

Antibodies Antibody Caspase 8 Cyclin d1 Dilution Electrophoresis Gapdh Immobilon p Membrane Poly a Protein Rabbit Sds page Src 1 Tnfα Vegf Western blot analysis

Corresponding Organization : National Institutes of Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Phosphorylation levels of Src (Y416), Rb (S780), and IkBα (S32/36)
Protein levels of total Src, total Rb, total IkBα, caspase 8, GAPDH, VEGF, TNFα, and cyclin D1

control variables

Protein sample amount (20 μg) loaded for SDS-PAGE
Antibody dilutions (e.g., 1:500, 1:1000) used for Western blot analysis
GAPDH used as a loading control

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

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