Western Blot Analysis of Inhibitor-Treated Cells

Western blot analysis of the inhibitor-treated cells was performed using a standardized protocol [22 (link)]. The primary human antibodies used in these analyses included NF-κB (p65), Smo, p53, p21, Nestin and β-actin (Santacruz, CA), IκBα, phospho-IκBα, phospho-NF-κB (p65), Bcl-2, S6K, phospho-S6K, 4E-BP1, phospho-4E-BP1, PLK1 and N-Myc (Cell Signaling Technology, MA) and cyclin D1, Bcl-2, and CD133 (BD Biosciences, CA). Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Santacruz, CA) and visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, IL).

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Chaturvedi N.K., McGuire T.R., Coulter D.W., Shukla A., McIntyre E.M., Sharp J.G, & Joshi S.S. (2016). Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan. Oncotarget, 7(12), 15215-15229.

Publication 2016

Nestin β-actin phospho-IκBα phospho-NF-κB (p65), Bcl-2 phospho-S6K 4E-BP1 phospho-4E-BP1 cyclin D1 peroxidase-conjugated secondary antibodies

4e bp1 Actin Antibodies Bcl 2 Cells Chemiluminescence Cyclin d1 Human Nestin Nf κb p65 Peroxidase Phospho Plk1 Western blot analysis

Corresponding Organization : University of Nebraska Medical Center

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Variable analysis

independent variables

Inhibitor treatment

dependent variables

Levels of NF-κB (p65)
Levels of Smo
Levels of p53
Levels of p21
Levels of Nestin
Levels of β-actin
Levels of IκBα
Levels of phospho-IκBα
Levels of phospho-NF-κB (p65)
Levels of Bcl-2
Levels of S6K
Levels of phospho-S6K
Levels of 4E-BP1
Levels of phospho-4E-BP1
Levels of PLK1
Levels of N-Myc
Levels of cyclin D1
Levels of CD133

control variables

Standardized western blot protocol [22]

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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