Phosphoinositide 3 kinase pi3k

Fucoidan (Undaria pinnatifida) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, penicillin-streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich. Antibodies against Bax, Bcl-2, β-actin, Akt, phospho-Akt (Ser473), caspase-9, phospho-PARP, phosphoinositide 3-kinase (PI3K), extracellular signal regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), P38 and rabbit lgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell lysis buffer and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The DeadEnd™ fluorometric terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay kit was purchased from Promega (Madison, WI, USA).

Proteins were isolated from cells and placental tissues by utilizing the RIPA lysate (Sigma, USA), and the concentrations of protein was measured by a BCA protein assay kit (Beyotime, China). After SDS-PAGE separation, the proteins were blotted onto PVDF membranes. After blocking with 5% skimmed milk at oom temperature for 1 h, the membranes were incubated with primary antibodies PGF (Abcam, UK), Phosphoinositide 3-kinase (PI3K; #4249, Cell Signaling Technology, USA), p-PI3K (ab278545, Abcam, UK), β-actin (ab8226, Abcam, UK), protein kinase B (AKT; #4685, Cell Signaling Technology; USA) and p-AKT (#4060, Cell Signaling Technology, USA) at 4°C overnight. Till next day, the membranes were washed twice, secondary antibodies which were labeled by enzyme and diluted were added, and incubated at room temperature for 1 h. The color development was done by chemiluminescence, and the results analysis was performed by gel imaging system. β-actin was selected as an internal reference protein.