Western Blot Analysis of Cellular Signaling Pathways

Western blot assays were performed as previously described (24 (link)). Briefly, proteins (20 μg/sample) were separated using SDS–polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes (Bio-Rad). Membranes were probed overnight with the following antibodies: phosphor-AKT (Ser 473), phosphor-AKT (Thr 308), AKT, N-MYC, phosphor-ACC (Ser 79), ACC, phosphor-GSK3b (Ser 9) GAPDH (obtained from Cell Signaling Technology), NAMPT, NAPRT (obtained from GeneTex) or α-tubulin (obtained from Abcam). Membranes were then washed, reacted with horseradish peroxidase–conjugated secondary antibodies and visualized using enhanced chemiluminescence (Thermo Scientific). Relative levels of NAMPT and NAPRT as well as quantitation of cleaved caspase 3 (active) and parp cleavage (indicative of caspase activation) were determined using Image J (imagej.nih.gov).

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Vallejo F.A., Sanchez A., Cuglievan B., Walters W.M., De Angulo G., Vanni S, & Graham R.M. (2022). NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth. Frontiers in Oncology, 12, 883318.

Publication 2022

nitrocellulose membranes phosphor-AKT (Ser 473), N-MYC NAMPT NAPRT α-tubulin enhanced chemiluminescence

Antibodies Caspase Caspase 3 Chemiluminescence Cleavage Gapdh Horseradish peroxidase Membranes Nampt Nitrocellulose Phosphor Proteins Sds polyacrylamide gel electrophoresis Western blot assays α tubulin

Corresponding Organization : University of Miami Health System

Other organizations : University of Utah Hospital, The University of Texas MD Anderson Cancer Center, Miami Children's Hospital

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Variable analysis

independent variables

Antibodies used for Western blot: phosphor-AKT (Ser 473), phosphor-AKT (Thr 308), AKT, N-MYC, phosphor-ACC (Ser 79), ACC, phosphor-GSK3b (Ser 9), GAPDH, NAMPT, NAPRT, α-tubulin

dependent variables

Relative levels of NAMPT and NAPRT
Quantitation of cleaved caspase 3 (active)
Quantitation of parp cleavage (indicative of caspase activation)

control variables

Protein amount loaded (20 μg/sample)
SDS–polyacrylamide gel electrophoresis for protein separation
Electroblotting to nitrocellulose membranes
Overnight incubation with primary antibodies
Washing of membranes
Incubation with horseradish peroxidase–conjugated secondary antibodies
Visualization using enhanced chemiluminescence

controls

Positive controls not mentioned
Negative controls not mentioned

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