D9e xp rabbit mab 4060

The cells were lysed in RIPA buffer (Sigma) with 1 mM NaF, 1 mM PMSF, 1 mM DTT,1mM Na₃VO₄with a complete and phosSTOP inhibitors (Sigma). The lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane by iBLOT Gel Transfer System (Invitrogen). The membrane was incubated with 5% nonfat milk w/v in TBS buffer (25 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Tween-20) for 1 h, and then reacted with the primary antibody in TBS buffer with 2.5 % nonfat milk or 5% BSA w/v for overnight by shaking at 4 degree. The appropriated second antibodies conjugated to HRP were used to detect the protein of interest via enhanced chemoluminescence (ECL). The following primary antibodies were used phosphor-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse mAb #9106, 1:2000; phosospho-Akt (Ser473) (D9E) XP rabbit mAb #4060, 1:2000; p44/42 MAPK (Erk1/2) antibody #9102, 1:1000; and Akt (pan) (11E7) rabbit mAb #4685, 1:1000 (all from Cell Signaling). The secondary antibody for rabbit primaries was goat anti-rabbit IgG (H + L)-HRP conjugate #7074, 1:3000; and for the mouse primaries was horse anti-mouse IgG, HRP-linked antibody #7076, 1:3000, (all from Cell Signaling).

Protein extracts from 24 hpf stage zebrafish embryos (n=80) were prepared as previously described [28 (link)], separated by 10% SDS-PAGE, and then transferred to PVDF membranes. The membranes were probed with either an anti-phosphorylated-AKT (Ser473) antibody (Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060) (1:1000) or an anti-AKT antibody (Akt Antibody #9272) (1:1000) from Cell Signaling Technology. Then, the membranes were incubated with a goat anti-rabbit HRP-conjugated secondary antibody (1:20,000). Signals were detected by incubation with a SuperSignal West Pico Chemiluminescent Substrate (Thermo) and ChemiDoc XRS (Bio-Rad).