Ab31488

With cold PBS flushing of the cultured cells, and then load in 1 x SDS buffer processing under 100°C for 10 minutes, samples were centrifuged at 12,000 rcf for 5 minutes; then, approximately 10 μl of protein was added to each electrophoresis channel lane and finally separated and shifted onto PVDF membranes. The obtained samples were sealed with skimmed milk mixture 5% for one hour at room temperature and then hatched with the primary antibody at 4°C for 8–12 h. Membranes that have been incubated with the primary antibody are rinsed with configured TBS-T buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) and finally incubated with horseradish peroxidase-conjugated secondary antibody. The antibodies were mouse anti-β-Tubulin (Cell Signaling, No 6181), rabbit anti-N-Cadherin (No 13116), and rabbit anti-E-Cadherin (No 3195) which were from Cell Signaling. The antibodies were rabbit anti-MCEMP1 (ab188572), rabbit anti-TLR4 (ab13556), human anti-NOD2 (ab31488), and rabbit anti-NFκB (ab32360) which were from Abcam.

The immunofluorescence assay was performed as described before^{15 (link)}. Antibody against NOD2 was purchased from Abcam (#ab31488, Cambridge, MA, USA), and the other antibodies were the same as used for western blot. The in vitro protein translation assay was performed by TNT Quick Coupled Transcription and Translation System (Promega, Madison, WI, USA) as previously described^{15 (link)}.