Cell Line Authentication and Kinase Inhibitor Assay

LC-2/ad cells were obtained from Sigma (cat no. 94072247), TPC1 cells obtained from R.E. Schweppe (17 (link)); H2228 cells obtained from J.D. Minna. HCC78-TAER were previously described (18 (link)). Cells were maintained in RPMI-1640 (Invitrogen) with 10% FBS at 37°C in a humidified 5% CO₂ incubator. Fingerprint analysis of cell lines was performed bi-annually by the Molecular Biology Service Center at the Barbara Davis Center for Diabetes at the University of Colorado Anschutz Medical Campus in Aurora, CO to ensure authenticity. Alectinib was provided by Chugai Pharmaceuticals. Ponatinib, cabozantinib, trametinib, gefitinib, afatinib, and foretinib were obtained from Selleck Chemicals. Pervanadate was generated by incubating hydrogen peroxide with 100 mM sodium orthovanadate in distilled water. Antibodies used were as follows: pEGFR Y1068 (D7A5), pEGFR Y1173 (53A5), total RET (D3D8R), pERK1/2 XP T202/Y204 (D13.14.4E), total ERK1/2 (L34F12), pAKT S473 XP (D9E), total AKT (40D4), and pSHC1 Y239/Y240 (2434) from Cell Signaling; pTYR (4G10 Platinum), GAPDH (6C5) and GAPDH (ABS16) from Millipore; pRET Y1062, α-tubulin (TU-02) and NRAS (F155) from Santa Cruz Biotechnology.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Nelson-Taylor S.K., Le A.T., Yoo M., Schubert L., Mishall K.M., Doak A., Varella-Garcia M., Tan A.C, & Doebele R.C. (2017). Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling. Molecular cancer therapeutics, 16(8), 1623-1633.

Publication 2017

LC-2/ad cells RPMI-1640 Ponatinib cabozantinib trametinib gefitinib afatinib foretinib pEGFR Y1068 pEGFR Y1173 pERK1/2 XP T202/Y204 NRAS (F155)

Afatinib Alectinib Antibodies Cabozantinib Cell lines Cells Diabetes Erk1 Foretinib Gapdh Gefitinib Hydrogen peroxide Nras Orthovanadate Pervanadate Pharmaceuticals Platinum Ponatinib Sodium Trametinib α tubulin

Corresponding Organization :

Other organizations : University of Colorado Denver

Top 5 similar protocols

Variable analysis

independent variables

Alectinib
Ponatinib
Cabozantinib
Trametinib
Gefitinib
Afatinib
Foretinib
Pervanadate

dependent variables

Cell responses (not explicitly specified)

control variables

RPMI-1640 media with 10% FBS
Incubation at 37°C in a humidified 5% CO2 incubator
Fingerprint analysis of cell lines performed bi-annually to ensure authenticity

positive controls

None specified

negative controls

None specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!