Ab2386

Protein lysates from Scr and shRNA TG2 expressing neurons were collected with RIPA buffer supplemented with protease inhibitors 6 days after transduction. Protein concentration of the samples was determined by BCA assay. Samples were diluted in collection buffer and 5× reducing stop buffer, boiled and resolved in a 12% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in tris buffered saline with Tween20 (TBS-T) (20 mM Tris base, 137 mM NaCl, 0.05% Tween-20) for one hour at room temperature. After blocking, the membranes were incubated overnight at 4°C with primary antibodies against serpinE1 (1:1,000, ABclonal (www.abclonal.com), A6211), vinculin (1:1,000, Bioss Antibodies (www.biossusa.com), bs-6640R), phospho (pY397) focal adhesion kinase (FAK) (1:1,000, Bioss Antibodies, bs-3159R), TG2 (clone CUB7402, 1:5000, Abcam (www.abcam.com), ab2386, or TGMO1, 1:5000 rat monoclonal antibody (Song et al., 2013)), tubulin (1:5000, Cell Signaling Technology (www.cellsignal.com), 2125S) or GAPDH (1:5,000, MilliporeSigma (www.emdmillipore.com), MAB374) in blocking buffer. Membranes were later washed three times with TBS-T and incubated at room temperature for one hour with HRP-conjugated secondary antibody. The membranes were washed with TBS-T and visualized using an enhanced chemiluminescence reaction.