Fig. 2.

ZEB1 interacts with the NuRD complex. Co-immunoprecipitation of Flag-ZEB1 in HEK/293 Flp-In (a), or co-IP of GFP-ZEB1 in the murine cell line 344SQ (5% input indicates whole cell lysate) (b). Blots for HDAC1 and ZEB1 (Flag-tagged or GFP-tagged) confirm ZEB1 interacts with HDAC1 and MTA1. c Validation of ZEB1/NuRD complex interaction by proximity ligation assay (PLA). Human and murine NSCLC cell lines were fixed, permeabilized, blocked, and probed with species specific primary antibodies directed against ZEB1 and NuRD complex members. PLA was performed utilizing the Duolink In Situ Red Starter kit (Sigma). Representative PLA signal and nuclear staining (DAPI) in the human NSCLC cells H157 are shown. Red signal signifies interaction between ZEB1 and HDAC1. Scale bars represent 100 µm. d Quantification of mean PLA signal per cell in various murine and human cell lines; standard deviation n = 5. Results from gel filtration conducted in e 344SQ and f H157 showing ZEB1 elutes in fraction number 25–35, corresponding with a megadalton-sized complex. Blots of the NuRD complex members CHD4 and HDAC1 confirm that the NuRD complex members concurrently reside within fractions 25–35. Superose column standards: F47/48 = 669 kDa, F51 = 440 kDa, F58 = 158 kDa, F61 = 44 kDa