ActA was conjugated through a heterobifunctional linker LC-SMCC (#22362; Thermo Scientific/Pierce, Rockford, IL), to poly-D-lysine (#P0296; Sigma Aldrich, St . Louis MO) labeled with ∼6 8 molecules of Bodipy FL-X-SE (#D6102; Molecular Probes) per peptide.
Cells were resuspended in PBS and stained with Live/Dead fixable Violet or Aqua exclusion dye (Molecular Probes, Invitrogen) per manufacturer instructions to identify viable cells.
Fractions were estimated to be at least 95% pure using SYPRO orange staining (molecular probes S5692, per protocol, sensitive to 5 ng of protein) with phosphoimager detection.
Labeling of the endoplasmic reticulum and Golgi apparatus was performed using C6-Ceramide or Endotracker, (Invitrogen, Molecular Probes) as per manufacturer's instructions.
PBMCs were then stained with 0.5 μg/mL of Cell Trace Oregon Green 488 (Molecular Probes, Invitrogen) per 1 × 10 cells as previously described [ 50].
Slides were stained with γ-H2AX antibody (Cell Signaling, Danvers, MA, USA) and with Alexa Fluor-488-labeled Fab2 (Molecular Probes) as per manufacturer's instructions.
Confluent MDA-MB-231 and ZR 751 cells grown in eight-well chamber slides were left untreated or pretreated with different concentrations of MβCD for one hour at 37°C, washed and labeled for lipid raft marker flotiilin or with GM1 (lipid raft labeling kit, Molecular Probes) as per the manufacturer's instructions.
It is also apparent in the molecular probe experiments [11].
Mouse anti-HA tag antibody (Abcam) and Alexa dye-conjugated phalloidin and secondary antibodies (Molecular Probes) were used per manufacturer's instructions.
Nitric oxide was quantitated by using Griess Reagent Kit for Nitrite Determination (Molecular Probes, USA) as per manufacturer's instructions.
