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. 2016 Jun 22;35(15):1656–1676. doi: 10.15252/embj.201694401

Figure EV2. C9orf72 interacts with the ULK1 initiation complex.

Figure EV2

  1. HeLa cells were transfected with FLAG‐FIP200, Myc‐C9orf72S and Myc‐C9orf72L or co‐transfected with FLAG‐FIP200 and either Myc‐C9orf72S or Myc‐C9orf72L as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with both anti‐FLAG and anti‐Myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = FLAG‐FIP200: 18, Myc‐C9orf72S: 22, Myc‐C9orf72L: 18, FLAG‐FIP200 + Myc‐C9orf72S: 18, FLAG‐FIP200 + Myc‐C9orf72L: 17). Scale bar = 20 μm.
  2. HeLa cells were transfected with HA‐ULK1, Myc‐C9orf72S and Myc‐C9orf72L or co‐transfected with HA‐ULK1 and either Myc‐C9orf72S or Myc‐C9orf72L as indicated. Transfections were laced with mVenus to enable identification of transfected cells for analysis (green). Transfected cells were probed with both anti‐HA and anti‐myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = HA‐ULK1: 20, Myc‐C9orf72S: 21, Myc‐C9orf72L: 20, HA‐ULK1 + Myc‐C9orf72S: 22, HA‐ULK1 + Myc‐C9orf72L: 20). Scale bar = 20 μm.
  3. HeLa cells were transfected with Myc‐ATG13, EGFP‐C9orf72S and EGFP‐C9orf72L or co‐transfected with Myc‐ATG13 and either EGFP‐C9orf72S or EGFP‐C9orf72L. The ATG13 transfection was laced with mVenus to enable detection of transfected cells for analysis (green). Transfected cells were probed with both anti‐EGFP and anti‐myc antibodies and processed for PLA. PLA proximity signals per cell (red) were determined (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ****P ≤ 0.0001; N (cells) = ATG13: 11, EGFPc2‐C9orf72S: 11, EGFPc2‐C9orf72L: 10, Myc‐ATG13 + EGFPc2‐C9orf72S: 11, Myc‐ATG13 + EGFPc2‐C9orf72L: 11). Scale bar = 20 μm.