Phospho hsl ser660

For staining of lipid droplets, we used HCS LipidTOX following the manufacturer’s instructions (Invitrogen). For live imaging of cells as described for fatty acids up-take we treated cells at day 5 of differentiation with BODIPY (10 μM) C₁₂ (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) (Invitrogen). In addition, to measure cell growth cells were stained with Incucyte NucLight Rapid Red Reagent for nuclear labeling following the manufacturer’s instructions (Sartorius). Those experiments were monitored using Incucyte live cell analysis system. For immunostaining 7 days post differentiation, cells were plated on coverslips for 24 hr and following fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, rinsed with PBS, and permeabilized with 0.2% Triton X−100 in PBS for 5 min at room temperature. Blocking was done with normal goat serum for 1 hr. Following primary antibody phospho-HSL (Ser660) 1:100 (Cell signaling) was added for overnight incubation. All samples were then incubated in secondary antibody 1: 1000, Alexa Fluor 488 conjugate (Abcam), and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen).

Western blotting was carried out as described previously (27 ). Both αTubulin and Ponceau were used as loading controls. All quantitative data were calculated using Ponceau control. αTubulin is presented as a control in representative images. The following primary antibodies used in this study were obtained from Cell Signaling Technology: PPAR-γ (CAT#: 2443), SCD1 (CAT#: 2438), FASN (CAT#: 3180S), ACC (CAT#: 3662), phospho-ACC Ser-79 (Cat#: 3661), ATGL (CAT#: 2439), phospho-HSL Ser-660 (CAT#: 4126), phospho-HSL Ser-563 (CAT#: 4139), phospho-HSL Ser-565 (CAT#: 4137), HSL (CAT#: 4107), AKT (CAT#: 9272), phospho-AKT Ser-473 (CAT#: 9271), and phospho-AKT Thr-308 (CAT#: 9275). The primary antibodies obtained from Abcam were DGAT2 (CAT#: 237613) and αTubulin (CAT#: ab7291). Other primary antibodies include SREBP1 (CAT#: 2215, Novus Biologicals) and PEPCK1 (CAT#: 10004943, Cayman Chemical). The secondary antibodies used were Goat Anti-Rabbit IgG (H + L)-HRP conjugate (CAT#: 1706515, Bio-Rad Laboratories), and Goat Anti-Mouse IgG (H + L)-HRP conjugate (CAT#: 1706516, Bio-Rad Laboratories).