Fig 13. Interaction between ABHD5 and ATGL depends on ABHD5 residues R297 and G326.

We transduced Lunet N hCD81 cells to co-express HA-tagged ABHD5 constructs and ATGL (WT or S47A) and harvested the cell lysates 48 hours post-transduction. We incubated the lysates on an anti-HA resin and detected ABHD5 and ATGL with specific antibodies by Western blot (mouse anti-ABHD5 and rabbit anti-ABHD5) in the inputs and the eluates. Note that roughly 5% of the inputs were loaded on the gels. (a) Immunoprecipitation of ATGL and ATGL S47A with HA-tagged ABHD5, with or without oleic acid induction of the cells. When indicated, we treated the cells overnight with 100 μM oleic acid before harvesting the lysates. (b) Three more examples depicting the co-immunoprecipitation of ATGL S47A together with HA-tagged ABHD5, in oleic acid-treated cells, in different experiments. Note that in two additional experiments, the interaction was under the detection limit. (c) Co-immunoprecipitation assay with the Chanarin-Dorfman syndrome mutant Q130P and the ABHD5 TBLC mutant. (d) Co-immunoprecipitation assay with the ABHD5 mutants of the predicted ATGL interface or with the ABHD4/5 chimeras. (c, d) Since the TBLC mutant and the ABHD4 constructs are not recognized by the anti-ABHD5 antibodies ([30] and Fig 11B), we also stained the membranes with an anti-HA antibody (rabbit) after the initial detections with the anti-ABHD5 and anti-ATGL antibodies.