Figure 6
LDAH decreases the levels of ATGL. (A) ATGL levels in Flag or Flag-LDAH transfected HEK293 during TAG hydrolysis as described in Fig. 5B. (B) Flag or Flag-human LDAH (hLDAH) were transfected to HEK293 cells, and cells were cultured with or without OA (360 μM) for 24 h. ATGL was detected using αnti-ATGL antibody. (C) Flag or Flag-LDAH were transfected to HEK293 cells. Cells were stimulated with OA for 24 h, and HSL, PLIN1, PLIN2, PLIN3 and ATGL were detected by immunoblotting with specific antibodies. Anti-actin antibody was used for loading control. Differentiated 3T3L1 adipocytes (+) were used for expression control. (D) Non-target or hLDAH siRNA were transfected to HEK293 cells. After 48 h OA (360 μM) was added to the culture media for 24 h. The expression levels of hLDAH and ATGL were determined by immunoblotting. (E) Flag-ATGL and/or LDAH were transfected to HEK293 cells as indicated. 24 h after transfection cells were treated with OA (360 μM for 24 h) followed by MG132 for 8 h as indicated in the figure. Flag-ATGL was pulled-down with an anti-Flag M2, and immunoblots were performed with anti-ubiquitin, anti-Flag, and anti-LDAH antibodies.
