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. 2020 Jun 11;39(15):e103649. doi: 10.15252/embj.2019103649

Figure 2. CALCOCO1 binds directly to ATG8 family proteins with preference for the GABARAP subfamily.

Figure 2

  1. GST pull‐down binding assay of in vitro transcribed/translated 35S‐Myc‐CALCOCO1 with recombinant GST‐tagged ATG8 family proteins. GST and GST fusion proteins were visualized by Coomassie Brilliant Blue staining (bottom panel), and the co‐precipitated Myc‐CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR.
  2. GST pull‐down assay of transiently transfected Myc‐CALCOCO1 from HEK293 cell extracts with recombinant GST‐tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co‐precipitated Myc‐CALCOCO1 detected by immunoblotting with anti‐Myc antibody (upper panel).
  3. CALCOCO1 deletion constructs used to map the ATG8 interactions. The red X indicate a point mutation or deletion of the LIR motif. Constructs with no or very weak interaction are indicated in orange.
  4. D‐H GST pull‐down assays of indicated in vitro transcribed/translated 35S‐Myc‐CALCOCO1 constructs with indicated recombinant GST‐tagged ATG8 family proteins. Precipitated GST and GST fusions and co‐precipitated Myc‐CALCOCO1 constructs were analyzed as in (A).
  5. GST pull‐down assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST‐tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co‐precipitated GABARAP with anti‐GABARAP antibody (upper panel).