Ab83354

HepG2 cell lysates were resolved in 4% to 12% gradient polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against ASM (rabbit polyclonal, Abcam, ab83354) and β-actin (mouse monoclonal, Santa Cruz Biotechnology, sc47778). Primary antibodies were followed by the appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich) and chemiluminescent Western blotting substrate (GE Healthcare) and processed on a ChemiDoc MP system (Bio-Rad Laboratories).

We employed a stringent RIPA formulation of 500 mM NaCl; 10 mM Tris–HCl, pH 8; 0.1% SDS; 5 mM EDTA, pH 8; 10× protease inhibitor (Complete tablet, Roche) to solubilize lipid rafts for subsequent immunoprecipitation experiments. Briefly, buoyant fractions, taken from the discontinuous gradient centrifugation, were pooled and incubated with the same volume of RIPA buffer on ice for 15 min. Resulting extracts were pre-cleared by rocking with 1 mL of a 5% slurry of RIPA equilibrated Protein A beads (CL-4B; Amersham Pharmacia, Uppsala, Sweden) for 4 h at 4 °C. After removal of the precipitate, the resulting supernatant was subjected to IP/Western blot assays. The following Abs were employed: α-CD19 (clone 6D5, Dako), α-ARID3A polyclonal Ab (produced in-house; [19 (link)]), α-IgM (BD Pharmingen), α-V5 (Sigma), α-Raftlin (graciously provided by Dr. Akihiko Yoshimura; 21), α-SUMO-1 (Sigma), anti-TFII-I (kindly provided by Dr. Carol Webb; [18 (link)]), pan α-actin (rabbit origin, Cytoskeleton, Inc.), α-Acid sphingomyelinase (SMPD1; ab83354, Abcam), goat α-EZRIN (sc-6407; Santa Cruz)

Transfected lung fibroblasts were fixed with 4% paraformaldehyde solution. After permeabilization and blocking treatment, cells were incubated with primary antibody against mouse α-ASM (1:500, # ab83354, Abcam) overnight at 4 °C. Next, sections were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse in dark. Finally, cells were stained with diamidino-2-phenylindole (DAPI; 1:1000; Beyotime, Nanjing, China) for 15 min. Data were acquired and analyzed using a fluorescence microscope (Nikon 80i, Tokyo, Japan).