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. 2023 Apr 20;18(4):e0280484. doi: 10.1371/journal.pone.0280484

Fig 6. AMD CIDEC variants in the CIDE-N domain decrease dimerization affinity and all four variants decrease binding to effector partners PLIN1, AS160 and RAB8A.

Fig 6

(A) 3xflag-tagged CIDEC wild-type (WT) was co-transfected with the indicated GFP-tagged CIDEC variants in HEK 293T cells. GFP alone was used as negative control. 3xflag-tagged CIDEC WT was immuno-precipitated (IP) using anti-Flag and pulled-down proteins were immuno-blotted (IB) with anti-GFP and anti-Flag. Total cell lysate was immunoblotted with anti-GFP to control for CIDEC-GFP expression levels. (B-E) HEK 293T cells were co-transfected with 3xFlag-CIDEC WT, E186X or AMD variants, and either PLIN1-mCherry (B), AS160-GFP (C) or RAB8A-mCherry (D). After immunoprecipitation (IP) of the 3xFlag-CIDEC, pulled-down proteins were probed with anti-mCherry or anti-GFP, and anti-Flag. Co-transfection with mCherry or GFP alone was used as negative controls. Total cell lysates were immunoblotted (IB) with anti-mCherry or anti-GFP to control for PLIN1, AS160 and RAB8A expression levels. (E) Representative fluorescence images of 3T3-L1 pre-adipocytes lipid droplets containing CIDEC-GFP wild-type (WT) or variants and RAB8A-mCherry. Scale bar: 2 μm.