Figure 10.

TP53INP2 interaction with GABARAP, GABARAP-like2, and LC3 proteins is dependent on autophagy. (a–c) Relative quantification of cytoplasmic TP53INP2-positive dots in autophagy-induced cells, in the presence or absence of wortmannin. HeLa cells were cotransfected with TP53INP2-EGFP and GABARAP-ERFP (a), GABARAP-like2-ERFP (b), or LC3-ERFP (c) and treated with EBSS (starvation) in combination with wortmannin (d–f). TP53INP2-pHRluc-C construct was used as BRET donor, and different amounts of GABARAP-pEYFP-C, GABARAP-like2-pEYFP-C or LC3-pEYFP-C fusion proteins were used as BRET acceptors. Cells were transfected in 12-well culture plates, and 1 day later, they were harvested and distributed in 96-well microplates. On the following day, the Rluc substrate coelentherazin-h was added 15 min before reading. Data are expressed as BRET ratio as described in Materials and Methods. (d) To study the interaction between TP53INP2 and GABARAP, GABARAP-like2, or LC3, cells were transfected with the BRET donor (TP53INP2-pHRluc-C) and BRET acceptors (GABARAP-EYFP, GABARAP-like2-EYFP, or LC3-EYFP) constructs, and autophagy was induced by starvation (e) or inhibited by wortmannin treatment (f). BRET values are expressed in milli-BRET units. Data are expressed as the mean ± SD of results from three independent experiments (*p < 0.05).