Skip to main content
. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: FEBS J. 2017 Nov 13;284(24):4216–4232. doi: 10.1111/febs.14301

Figure 5. The rabbit reticulocyte lysate subunit interaction screen identifies new interactions.

Figure 5

(a) Summary of all the interaction crosses performed. The ‘MTA only’ label for the MBD3-MTA2N+HDAC1 cross indicates that MBD3 interacted only with MTA2N but not HDAC1. (b) Western blots of positive interactions observed in (a) are shown here. Five pairs of interactions were observed: MTA2-MTA2, MBD3-MTA2, RBBP4-MTA2, GATAD2B-MBD3 and CHD4C1C2-GATAD2B. Bait proteins have been denoted with * while the prey proteins, if observed in the pulldown lane (PD), have been denoted with +. For the MTA2-MTA2 interaction, the corresponding αHA-only blot has been provided to show that the HA-tagged MTA2 bait protein has been pulled down. (c) Summary of all the pulldowns performed with the fragments of MTA2, CHD4 and GATAD2B. (d) Corresponding western blots of all tested interactions as summarised in (c). RBBP4 was observed to interact with MTA2C and GATAD2B with CHD4C2. The interaction between GATAD2B and CHD4C2 was then further narrowed down to the residues 276–473 of GATAD2B (construct GATAD2Be). Bait proteins have been denoted with * while the prey proteins, if observed in the pulldown lane (PD), have been denoted with +. Where the bait and prey proteins are of similar molecular masses, the corresponding αHA-only blot is provided. (e) Immunoprecipitations using FLAG-GATAD2Af (339–633) as bait in transfected HEK293 cells. The western blots were incubated with the antibodies indicated on the left. GATAD2Af is able to pull down CHD4 but not other NuRD components. IgG: negative control using IgG for the immunoprecipitation. Note that in all cases, the relative intensities of bait and prey bands do not correlate with the concentrations of the relevant proteins because of the different activity of the anti-FLAG and anti-HA antibodies.