Dy light 660 anti rabbit secondary antibody

The PSD95 immunoprecipitation assay was probed with PSD-95 rabbit primary antibody (1:1000, Cell Signaling) to check for presence of PSD-95 pulled down. Equal amounts of sample were loaded to probe interaction of PSD-95 with GluR2 (Millipore), GluR1, and ILK with rabbit primary antibodies (1:1000, Cell Signaling). Whole hippocampal protein lysates were probed for BDNF or proBDNF, and GSK3β to total GSK3β, using their respective primary antibodies at 1:1000 (Cell Signaling). All blots were probed with Dy-Light 660 anti-rabbit secondary antibody (1:10000, Thermo Scientific) using a Fuji FLA 5100 scanner. They are presented as means ± SEM. Significance was determined using a two-tailed Student's t-test.

ILK activity was determined in hippocampal tissue homogenates using an immune complex kinase assay [21 (link)–24 (link)]. Briefly, tissue lysates were pooled and incubated with 1:50 anti-ILK mAb (cell signaling). The resulting immune complexes were washed three times in kinase reaction buffer, followed by incubation with 1 μg inactive Akt and ATP (final concentration: 200 μM) in 50ul kinase reaction buffer for 1 h at 30°C. The reaction products (supernatant) were resolved on SDS/PAGE. The beads were then processed as described in IP assay for ILK immunoblot. Membranes were probed with antiphospho-Akt (ser⁴⁷³) mAb (Cell Signaling Technology). The blot was developed using Dy-Light 660 anti-rabbit secondary antibody (1:10000, Thermo Scientific) using a Fuji FLA 5100 scanner. The data are presented as means ± SEM. Significance was determined using a two-tailed Student's t-test.