Anti cea

The SGECs on coverslips were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 (Sigma) for 20 min at room temperature. Subsequently, 10% FBS was used to block non-specific binding. The SGECs were then incubated with primary antibodies at 4°C overnight. The antibodies that were used were the following: anti-CD7, anti-CD8, anti-CD14, anti-CD15, anti-CD18, anti-CD19, anti-CEA and anti-LGR5 (Abcam). The cells were then incubated with Alexa Fluor 488-conjujated anti-mouse/anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.) for 1 h at room temperature. Nuclei were stained with Hoechst 33342.

Gold gate electrode is functionalized with receptors (antibody and aptamer) by the strong S-Au covalent binding. Disulfide bonds in the hinge region of IgG molecule is cleaved using moderate reducing agent such as 2-mercaptoethylamine (2-MEA) resulting in thiol terminated half IgG molecules which can self-assemble on to the gold gate electrode. Monoclonal antibodies (anti-CEA and anti-NT-pro BNP) are obtained from Abcam and 2-MEA from Sigma Aldrich. IgG and MEA mixture is incubated in room temperature on the device for 1.5 hours and at 4 °C for 12 hours. Unbound half-IgG is washed away using PBS before detection of proteins. Thiol modified oligonucleotides specific to HIV-1 RT and CRP are purchased from Genomics. Original aptamer solution is diluted in TE buffer containing tris (2-carboxyethyl)phosphine (TCEP) in the molar ratio 1:1000 and heated up to 95 °C to separate the single stranded DNAs. After the mixture is cooled down, 5 µl is dropped on the device and incubated at room temperature for 24 hours. The device is thoroughly washed in TE buffer and PBS prior to measurement.

The ALOX15, USP7 and hnRNPA1 expression were assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4 °C overnight with anti-ALOX15 (1:1000, Santa cruz), anti-hnRNPA1 (1:1000, Santa cruz), anti-CD63 (1:2000, Abcam), anti-TSG101 (1:1000, Santa Cruz), anti-Alix (1: 1000, Santa Cruz), anti-ubiquitin (1:1000, Santa Cruz), anti-α-SMA (1: 1000, Abcam), anti-FAP (1: 1000, Abcam), andti-FSP1 (1: 1000, Abcam), anti-CEA (1:1000, Abcam), anti-CK-18 (1:1000, Abcam) and anti-GAPDH (1:3000, Santa Cruz) antibodies respectively.