Distinguishing parallel from series circuits with a double knockdown procedure in human cell lines

Angela M Gocher-Demske; Shuhang Dai; Arthur M Edelman

doi:10.1016/j.xpro.2022.101890

Distinguishing parallel from series circuits with a double knockdown procedure in human cell lines

STAR Protoc. 2022 Dec 16;3(4):101890. doi: 10.1016/j.xpro.2022.101890. Epub 2022 Nov 30.

Authors

Angela M Gocher-Demske¹, Shuhang Dai², Arthur M Edelman²

Affiliations

¹ Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Tumor Microenvironment Center, University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center, Pittsburgh, PA 15232, USA. Electronic address: agocher@pitt.edu.
² Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14203, USA.

Abstract

Signaling cascades can act in series or in parallel. Here, we describe a convenient and robust protocol for dual, sequential knockdown of two proteins using RNA interference. We detail the steps for a quantitative mapping of signaling circuitry. We used this approach to study kinases in human ovarian cancer cells, but the protocol can be applied to many other posttranslational modifications. For complete details on the use and execution of this protocol, please refer to Gocher et al. (2017).¹.

Keywords: Cell Biology; Molecular Biology; Protein Biochemistry; Signal Transduction.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural

MeSH terms

Cell Line
Female
Humans
Ovarian Neoplasms* / genetics
Protein Processing, Post-Translational
RNA Interference
Signal Transduction

Grants and funding

F32 CA247004/CA/NCI NIH HHS/United States