Mouse Anti Human Ras-related C3 Botulinum Toxin Substrate 1 Hybridoma [DQUD-SBD2-3]

Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation assay (PLA). PLA is a specific and sensitive method that allows the observation of interacting proteins by target-specific antibody detection coupled to rolling circle amplification. ELMO1 is the regulatory subunit of ELMO1-dedicator of cytokinesis 180 (DOCK180) bipartite Rac1 guanine nucleotide exchange factor (GEF) which adopts a closed autoinhibitory conformation via an intramolecular interaction of its N-terminal ELMO inhibitory domain (EID) and C-terminal ELMO autoregulatory domain (EAD). In the assay, PLA signals are detected in cells transfected with ELMO1(1-315) and ELMO1(315-727) fragments. Moreover, overexpression of FE65, a neuronal adaptor which has been shown to disrupt ELMO1 intramolecular interaction, reduces the PLA signals of the two ELMO1 fragments significantly. Together, our results demonstrate that PLA can be employed for studying protein intramolecular interaction.

Estrogen Receptor alpha (ER alpha) affects the morphology of tumors, which is closely related to the biomechanical properties and the cytoskeletal proteins. In recent years, researchers have found that biomechanical properties and cytoskeletal proteins are closely related to the occurrence and development of tumors and that biomechanical properties can be used as markers for tumor development and drug resistance. The relationship between ER alpha expression status and biomechanical properties, cytoskeletal proteins is not known. In this study, we found that tamoxifen-resistant breast cancer cells (MCF-7/TamR) altered cell morphology and lacked of ER alpha expression during the process of the Tamoxifen resistance induction. To determine whether this change was influenced by ER alpha expression, we transiently constructed another ER alpha depleted model with ER alpha siRNA (MCF-7/ER alpha siRNA) and used atomic force microscope (AFM) to detect morphological and biophysical changes. The results indicated that the roughness and Young's modulus of ER alpha expression depleted cells were significantly increased, accompanied by rearrangement of the cytoskeletal proteins (F-actin, FLNA, alpha-tubulin) and the cytoskeletal regulatory protein Rho (Rac1, CDC42) decreased. Our results have demonstrated that ER alpha depletion affects the biomechanical properties of breast cancer cells, which are related to cytoskeletal protein rearrangement and Rho protein decreased. (c) 2020 Elsevier Inc. All rights reserved.