Immunoblot Analysis of Signaling Proteins

Whole-protein extracts from control or treated JIMT1 cells were obtained as previously described by cell scraping at 4 °C in a radioimmunoprecipitation assay buffer.^{20 (link)} Following electrophoresis and transfer to nitrocellulose membranes (Thermo Fisher Scientific, IB23001), the blots were incubated in 5% w/v bovine serum albumin (BSA, Sigma, A7030) in Tris-buffered saline buffer-Tween (TBS-T, Cell Signaling Technology, 9997S) and probed with mouse anti-β-actin 1:20 000 (Sigma, A1978), rabbit anti-HER2 1:800 (Abcam, ab131490), rabbit anti-pHER2 1:800 (Abcam, ab53290), rabbit anti-EGFR 1:1000 (Abcam, ab52894), rabbit anti-pEGFR 1:500 (Abcam, ab40815), rabbit anti-HER3 1:500 (Abcam, ab32121), rabbit anti-pHER3 1:2500 (Abcam, ab76469), rabbit anti-AKT 1:1000 (Cell Signaling, 9272), rabbit anti-pAKT 1:2000 (Cell Signaling, 4060), mouse anti-ERK 1:1000 (Thermo Fisher Scientific, 14-9108-82), rabbit anti-pERK 1:500 (Thermo Fisher Scientific, 700012), rabbit anti-CD44 1:1000 (Abcam, ab189524), and rabbit anti-CAV1 1:500 (Abcam, ab2910) antibodies. After the antibodies were incubated and washed, the membranes were incubated with IRDye800CW 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).

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Pereira P.M., Ragupathi A., Shmuel S., Mandleywala K., Viola N.T, & Lewis J.S. (2019). HER2-Targeted PET Imaging and Therapy of Hyaluronan-Masked HER2-Overexpressing Breast Cancer. Molecular pharmaceutics, 17(1), 327-337.

Publication 2019

TBS-T ab131490 ab53290 ab52894 ab40815 ab32121 rabbit anti-AKT rabbit anti-pAKT rabbit anti-pERK ab189524 ab2910 Odyssey Infrared Imaging System

Actin Antibodies Bovine serum albumin Buffer Cav1 Cd44 Cell Densitometric Egfr Electrophoresis Erk 1 Her2 Her3 Membranes Mouse Nitrocellulose Protein Rabbit Radioimmunoprecipitation assay Saline Tween

Corresponding Organization : Cornell University

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Variable analysis

independent variables

Control or treated JIMT1 cells

dependent variables

Levels of proteins (β-actin, HER2, pHER2, EGFR, pEGFR, HER3, pHER3, AKT, pAKT, ERK, pERK, CD44, CAV1)

control variables

Whole-protein extracts were obtained by cell scraping at 4 °C in a radioimmunoprecipitation assay buffer
Electrophoresis and transfer to nitrocellulose membranes
Membranes were incubated in 5% w/v bovine serum albumin (BSA) in Tris-buffered saline buffer-Tween (TBS-T)
Antibodies used for probing the membranes

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