HUVEC were grown to 80–90% confluency, suspended as above, and fractionated into their subcellular compartments using kits from Thermo Scientific (Logan, UT). The following antibodies (Cell Signaling, Danvers, MA) were used to define different subcellular fractions: rabbit anti-human HDAC2 (nuclear protein), rabbit anti-human histone-H3 (nuclear chromatin), and mouse anti-human vimentin (cytoskeleton). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (Cell Signaling) served as secondary antibodies.
For TM4SF1 pull-down assays, suspended HUVEC were pre-incubated with 8G4 or with an isotype-matched mouse IgG1 control antibody for 1 hour on ice, washed 3x with PBS to remove unbound antibody, and returned to culture for 4h at 37°C before cells were harvested for total protein extraction in a cell lysis buffer comprised of Tris-buffered saline (TBS), pH 7.0, protease/phosphatase inhibitor cocktails, and 0.1% Triton X-100 (Life Technology). Protein-G beads were then added to the lysates to pull down 8G4 (or control IgG). The 8G4 pull down fraction was then electrophoresed and immunoblotted with 8G4 that had been conjugated with HRP (Life Technology labeling kit).
For TM4SF1 pull-down assays, suspended HUVEC were pre-incubated with 8G4 or with an isotype-matched mouse IgG1 control antibody for 1 hour on ice, washed 3x with PBS to remove unbound antibody, and returned to culture for 4h at 37°C before cells were harvested for total protein extraction in a cell lysis buffer comprised of Tris-buffered saline (TBS), pH 7.0, protease/phosphatase inhibitor cocktails, and 0.1% Triton X-100 (Life Technology). Protein-G beads were then added to the lysates to pull down 8G4 (or control IgG). The 8G4 pull down fraction was then electrophoresed and immunoblotted with 8G4 that had been conjugated with HRP (Life Technology labeling kit).