Bullet blender homogenizer bbx24

Liver tissues and cultured macrophages were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) using a BBX24 Bullet Blender homogenizer (Next Advance Inc., Averill Park, NY) according to the manufacturer's instruction. The lysates were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes. Western blot analysis was carried out as reported previously by probing the blots with the indicated primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (Yang et al., 2013 (link)). The reactive bands were visualized using an ECL Plus Western Blotting kit (PIERCE, Rockford, IL) according to the manufacturer's instructions. Primary antibodies against CX3CR1 (1:1000), indoleamine 2, 3-dioxygenase (IDO, 1:1000), STAT6 (1:500), PPAR-γ (1:500), and β-actin (1:500) were obtained from Abcam (Cambridge, MA), whereas antibodies against iNOS (1:250) and Arg-1 (1:4000) were purchased from BD Pharmingen (Carlsbad, CA). The intensity of each reactive band was analyzed using the densitometry plugin ImageJ software (http://rsb.info.nih.gov/ij/).

Sterol-producing yeasts were kindly provided from the Riezman lab (Supplementary Table 5)^{35 (link)}. Competent cells of these strains were prepared using the Frozen EZ Yeast Transformation II Kit^TM (Zymo Research, Irvine, CA, USA). Starter cultures were grown in the SD-Leu medium at 30 °C overnight and then used to inoculate 25 mL SD-leu medium in triplicates in a shaking flask with an initial OD₆₀₀ of 0.2–0.4. Samples were harvested at 18 h and pelleted 3000 g for 5 min. Yeast cells were resuspended in 200 μL of TES buffer (50 mM Tris-HCl pH = 7, 600 mM sorbitol, 10 g/L bovine serum albumin, 1.5 mM β-mercaptoethanol) and homogenized with an equal volume of 0.5 mm glass beads in a BBX24 Bullet Blender® homogenizer (Next Advance, Troy, NY, USA) at setting 8 at 4 °C for 4 min. A total of 300 μL of TES buffer was added to the lysed cells, and 400–500 μL of the yeast lysate was transferred into a capped glass tube, followed by adding 1 mL chloroform immediately. The sample was vortexed for 1 min, and the organic phase was transferred into a new glass test tube and dried under a stream of air. The sample was resuspended in 100 μL methanol, centrifuged at 17,000 g for 10 min, and the supernatant was transferred into a LC/MS vial and stored at −20 °C until use.