Clone spm227

NIH cells (Fig. 2) were seeded onto cover slips placed in 24-well plates. After 24 h, the medium was changed to new medium containing 360 μM oleic acid. After 20 h, the cells were fixed with 2% formaldehyde for 10 min and stored at 4°C in PBS. For immunolabeling, the cells were treated with Avidin/Biotin Blocking kit (Vector Laboratories), and 1% BSA in PBS for 20 min. The cells were then incubated 2h at room temperature with mouse anti-vinculin (1:100, clone SPM227, Abcam), followed by 30 min biotinylated monovalent donkey anti-mouse IgG (1:200, Jackson ImmunoResearch) and 30 min with Atto425-conjugated streptavidin (1:200, ATTO-TEC). The cells were then incubated for 2h at room temperature with guinea pig anti-ADRP (1:400, Fitzgerald), rabbit anti-mouse syntaxin 5 (1:150, Synaptic System) and rat anti-mouse LAMP1 (1:100, clone 1D4B, Abcam) followed by 30 min incubation with goat anti-guinea pig AF488 (1:400, Life Technologies), goat anti-rabbit Cy3 (1:250, Jackson ImmunoResearch), mouse anti-rat PerCP-eFluor710 (1:100, Affymetrix) and phalloidin-Atto594 (5 μL/mL, Sigma-Aldrich); in the last 5 min, 3 drops of DAPI (2 μg/mL) was added to each well. All incubation steps contained 0.1% saponin and all washing steps contained 0.05% saponin for permeabilization. The cover slips were mounted onto microscope slides with Prolong Gold antifade reagent (Life Technologies).

Cells were lysed in 1% Triton X-100 lysis buffer (20 mM Tris-HCl at pH 8, 100 mM NaCl, 10% glycerol) for 30 min on ice. Cell lysates (20–100 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and immunoblotting using anti-vinculin antibody (1:5000: clone SPM227; Abcam) or anti-HA tag antibody (1:1000; clone 6E2; Cell Signaling Technology). Secondary antibody used was horseradish peroxidase-conjugated anti-mouse immunoglobulin G (1:5000; NA931V; GE Healthcare).