Protein Expression Analysis in PDX Models

Whole-protein extracts from PDXs were obtained in radioimmunoprecipitation assay buffer as previously described (25 (link)). After electrophoresis and transfer to nitrocellulose membranes (IB23001; Thermo Fisher Scientific), the blots were incubated in 5% w/v bovine serum albumin (A7030; Sigma) in Tris-buffered saline with polysorbate (9997S; Cell Signaling Technology) and probed with mouse anti-β-actin, 1:20,000 (A1978; Sigma); rabbit anti-HER2, 1:800 (ab131490; Abcam); rabbit anti-phosphorylated HER2 (anti-pHER2), 1:800 (ab53290; Abcam); rabbit anti-EGFR, 1:1,000 (ab52894; Abcam); rabbit anti-MET, 1:1,000 (ab51067; Abcam); and anti-phosphorylated MET (anti-pMET), 1:1,000 (ab68141; Abcam). After antibody incubation and washing, the membranes were incubated with the secondary antibodies IRDye 800CW anti-rabbit (925-32211) or anti-mouse (925-32210) IgG, 1:15,000 (LI-COR Biosciences), and imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences), followed by densitometric analysis using Fiji software (https://imagej.net/Fiji) (32 (link)).

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Pereira P.M., Norfleet J., Lewis J.S, & Escorcia F.E. (2021). Immuno-PET Detects Changes in Multi-RTK Tumor Cell Expression Levels in Response to Targeted Kinase Inhibition. Journal of Nuclear Medicine, 62(3), 366-371.

Publication 2021

IB23001 A7030 A1978 ab131490 ab53290 ab52894 ab51067 ab68141 Odyssey Infrared Imaging System

Actin Antibodies Antibody Bovine serum albumin Buffer Densitometric Egfr Electrophoresis Her2 Irdye 800cw Membranes Mouse Nitrocellulose Pdxs Polysorbate Protein Rabbit Radioimmunoprecipitation assay Saline

Corresponding Organization :

Other organizations : Memorial Sloan Kettering Cancer Center, Cornell University, Center for Cancer Research, National Cancer Institute, National Institutes of Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of proteins: β-actin, HER2, phosphorylated HER2 (pHER2), EGFR, MET, and phosphorylated MET (pMET)

control variables

Protein extraction method using radioimmunoprecipitation assay buffer
Electrophoresis and transfer to nitrocellulose membranes
Blocking conditions: 5% w/v bovine serum albumin in Tris-buffered saline with polysorbate
Primary antibody concentrations: mouse anti-β-actin (1:20,000), rabbit anti-HER2 (1:800), rabbit anti-pHER2 (1:800), rabbit anti-EGFR (1:1,000), rabbit anti-MET (1:1,000), and rabbit anti-pMET (1:1,000)
Secondary antibody concentrations: IRDye 800CW anti-rabbit or anti-mouse IgG (1:15,000)
Imaging system: Odyssey Infrared Imaging System
Densitometric analysis using Fiji software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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