1%. The lysates till were then centrifuged, aliquoted, and stored at 70 C until use. Protein concentration was Inhibitors,Modulators,Libraries determined by BCA assay. Each experiment was repeated at least two times. For western analysis, membranes were blocked with 5% BSA in the rinse buffer for 1 h, washed in rinse buffer for 10 min, and then incu bated with the respective primary antibody at the indi cated concentrations. The membranes were then washed and incubated with the appropriate horse radish peroxidase conjugated secondary antibody for 1 h at room temperature, washed for 10 min and four more cycles of 5 min, and treated with an enhanced chemiluminescence detection system. In some cases, when the signal was very weak or undetectable, we used ECL plus.

Effect of PI3K/Akt and MEK inhibitors, or Pertussis Toxin on Saposin Inhibitors,Modulators,Libraries C Activation of p42/44 MAPK Cells were grown in their respective complete culture media for 2 3 days, washed with PBS, incubated in their serum free media for 20 h, and then fresh basal media was added Inhibitors,Modulators,Libraries to all plates for an additional 4 h. Various inhibitors were added to the culture medium, just before treating cells with saposin C. We used 10% FBS as a positive con trol. Cells were lysed and 10g of clarified protein sam ples was subjected to SDS PAGE under reducing conditions. Phospho specific p42/44 antibody was used as the primary antibody and as a loading control. Filters were also probed or reprobed with anti p42/44 antibody. Addi tional tissue culture plates that had been treated with or without inhibitors were also Inhibitors,Modulators,Libraries tested for cell viability by trypan blue dye exclusion assay.

Saposin C and PI3K/Akt signaling pathway Cells were cultured up to 70% confluency in their com Inhibitors,Modulators,Libraries plete media and after washing with PBS, they were serum starved for 24 h, and then treated with 10% FBS or saposin C at 0. 1, 1 or 10 nM for 10 min. A representative tissue culture plate was also pretreated with the PI3K inhibitor before treating cells with saposin C. After preparation of the cell lysate, 15g of protein per sample was subjected to SDS PAGE under reducing conditions. Immunoblotting was performed using phospho specific Akt antibodies against serine 473 or threonine 308. A loading control was evaluated with anti Akt antibody. Each exper iment was performed in duplicate, and the assays were repeated three times. Immunoprecipitation and in vitro Akt kinase activity assay A non radioactive Akt kinase assay kit was used to determine whether saposin C treatment of cells under serum starvation stress would lead to Akt activation. For Akt kinase assays, cells were grown up to 70% confluency in their maintenance media, serum starved for 24 h, and then treated selleck Belinostat for 5 or 10 min in the presence or absence of saposin C at 0. 1, 1, or 10 nM.