Ctxb fitc

Cells were treated with phosphate-buffered saline (PBS) containing 0.53 mM ethylenediaminetetraacetic acid (EDTA), suspended in cold FACS buffer (0.1% foetal bovine serum in PBS), and then stained with either CTXB-FITC (12.5 μg/mL, SIGMA) for ganglioside M1 (GM1) or isotype immunoglobulin G (IgG)-FITC (Jackson) as a negative control for 30 minutes at 4 °C. Cells were washed with PBS and then examined via FACS analysis (FACSCalibur, BD). Untreated or simvastatin-treated cells were fixed with 4% paraformaldehyde, labelled with CTXB, and imaged with a Leica SP2 confocal microscope.

Immunofluorescence analysis of HA-STK4 protein in C4-2 cells was performed with modifications [27 (link)]. Briefly, cells were fixed with freshly prepared 4% PFA (paraformaldehyde that was prepared in PBS) for 30 min and permeabilized with 0.2% Triton-X-100 and incubated with anti-HA (Covance, 1:50) antibody overnight at 4°C. Cells were washed with PBS after each step. In addition, lipid rafts were labeled with CTxB-FITC conjugated (Sigma-Aldrich) as described [21 (link)]. Briefly, live cells were washed with cold PBS and incubated with CTxB-FITC (20 ng/ml, which was prepared in cold serum-free media) on ice for 30 min prior to fixation with 4% PFA. Alexa Fluor 532 conjugated anti-mouse (1:1000 dilution) was used to detect HA-STK4 signals in the cell. Slides were mounted with VectaShield containing DAPI (Vector Labs, H-1200). Immunofluorescence images were captured by microscopy (Zeiss 700) at 40x magnification with oil immersion.

Fibroblasts from the wild-type (WT) control and from GD patients carrying c.1226A>G (N370S)/RecNcil, c.1448T>C (L444P) and c.508C>T p. (R131C) mutations were plated in 12-well plates containing glass coverslips at a 30,000 cells/well density. Twenty-four hours after plating, the cells were fixed with 4% PFA, rinsed with PBS (+MgCl₂ 0.5 mM, +CaCl₂ 0.8 mM) and permeabilized with 0.075% Triton X. After rinsing with PBS and blocking with 4% BSA PBS, cells were incubated for 45 min with rabbit polyclonal anti-Glc 0.02% NaN₃ (RAS0010, Glycobiotech, Kükels, Germany, diluted at 1:250) or 10 µg/mL Cholera Toxin B subunit—Fluorescein isothiocyanate conjugate (CTXb-FITC, Sigma-Aldrich Merck, Burlington, MA, USA) primary antibodies to stain GlcCer and GM1, respectively. After washing the coverslips with PBS, cells were incubated for 40 min with the secondary antibody Alexa 568 anti-rabbit (Invitrogen, diluted at 1:500). The coverslips were then washed with PBS and water and mounted on a glass slide. Cell imaging was performed on a Nikon Eclipse TE300 C2 confocal microscope (Nikon, Tokyo, Japan) equipped with a Nikon 100× immersion oil objective (Apo Plan, NA 1.4), with Melles Griot (Argon 488 nm) and Coherent (Sapphire 561 nm) lasers. Emission filters for imaging were 514/30 nm and 595/60 nm.