Skip to main content
. 2020 Sep 22;39(22):e105098. doi: 10.15252/embj.2020105098

Figure EV5. linc‐MYH interacts with specific domains of INO80.

Figure EV5

  1. INO80 protein with functional domains labeled (NTD: N‐terminal domain, HSA: helicase‐SANT-associated domain, Snf2 ATPase: Snf2 ATPase domain). Numbers indicate positions in the sequence of the hINO80 protein (NP_060023.1).
  2. Pull‐down experiments using in vitro transcribed linc‐MYH RNA (AK079404) or control lincRNA to pull‐down human flag‐tagged INO80 protein (hINO80) or hINO80 fragments ectopically expressed in HEK293 cells. Proteins binding to linc‐MYH RNA were identified by Western blot using an anti‐FLAG antibody. The hINO80 protein binds to linc‐MYH as expected, but the interaction is lost following deletion of the NTD (ΔNTD) domain or after deletion of the NTD and HSA (ΔNTD ΔHSA) domains. Accordingly, we detect interaction of the NTD (NTD) domain and of the HSA (HSA) domain with linc‐MYH.
  3. Pull‐down experiment using in vitro transcribed linc‐MYH RNA (AK079404) to test its interaction with mouse V5‐tagged MCRS1 protein (MCRS1) ectopically expressed in HEK293 cells.
  4. Pull‐down experiments using the different in vitro transcribed exons of linc‐MYH splice variants (AK079404, AK010044) as well as four different controls to detect interaction with native INO80 protein from nuclear extracts of C2C12 myoblasts. The experiment was performed in duplicate; interacting proteins were identified by mass spectrometry. No INO80 was detected in controls; however, INO80‐specific peptides were detected to specifically interact with exon sequences of linc‐MYH. Exons labeled in blue indicate detection of the INO80 protein, exons labeled in red indicate failure to detect the INO80 protein, and the exon labeled in gray was not tested.
  5. Pull‐down experiments using different in vitro transcribed exons or different parts of the last exon of linc‐MYH along with FLAG‐tagged human INO80 from HEK293 cell lysates.