Figure 4
LDAH’s role in LD growth and TAG accumulation is related to its C-terminal region rather than to its catalytic sites. (A) Diagrams of point mutants of full-length LDAH used in the studies, including mutations of hystidine to alanine in predicted acyltransferase motifs, and of serine to alanine in the predicted hydrolytic motif. (B) TAG levels (top) and immunoblotting with anti-Flag antibody (bottom) in HEK293 cells overexpressing Flag-wild type or Flag-mutant LDAH, and loaded with OA (360 μM) for 24 h. *p < 0.05, **p < 0.005. (C) Diagram of predicted LDAH splicing variants. (D) mRNA expression of LDAH isoforms 1, 2, and 3 during 3T3L1 differentiation. (E) TAG levels in Flag-tagged LDAH isoform 1 and isoform 3 overexpressing cells stimulated with OA (360 μM) for 24 h. *p < 0.05. (F) TAG levels in Flag-tagged isoform 1 and isoform 2 overexpressing cells treated with OA (360 μM) for 24 h. *p < 0.05. Immunoblottings (bottom) were performed using a anti-Flag antibody. (G) LDAH isoform 1-GFP and LDAH isoform 2 -GFP were transfected to HEK293 cells. After 24 h cells were treated with OA (360 μM) for 24 h. LDAH was visualized by fluorescence microscopy (green) and LDs were stained with LipidTox (red).
