In fact, the molecular dynamics studies which describe a scissor like molecular

In these experiments, the cells were treated with 2. 5 100 uM PDMP and incubated for 72 h. After selec tion of optimal concentration, EL 4 cells were cultured for 6 days in the presence of 15 uM PDMP before the analysis of cytotoxicity induced by treatment with anti GD2 antibodies. Knockdown of supplier INNO-406 GM2 GD2 and GD3 synthases by siRNA siRNA for mouse GM2 GD2 or GD3 synthases were pur chased from Santa Cruz Inc. The cells were transfected with these siRNAs using lipophilic agent Lipofectamine 2000 according to the manufacturers instruc tions. Cells were harvested 48 h post transfection and further incubated with anti GD2 mAbs for 24 h followed by performing PI test.

Western blot supplier Lapatinib analysis Protein lysates of EL 4 cells were prepared using RIPA buffer, The proteins from cell lysate were fractionated in SDS PAGE, and were transferred onto nitrocellulose membranes using a semi dry transfer device V10 SDB, Membranes were further in cubated in blocking buffer for 1 h at RT, followed by incubation in primary anti GM2 GD2 synthase antibody for 1 h at RT in PBS supplemented with 0. 05% Tween 20, After washing several times with PBS T, the membranes were incubated for 1 h in HRP conjugated secondary antibody at RT, and then were washed four times with PBS T. The immunoreac tive proteins were visualized using the Metal Enchanced DAB Substrate Kit according to the manufacturers instructions. RNA isolation and cDNA synthesis Cells transfected with siRNA that target GM2 GD2 synthase or control cells were dissolved in 0.

5 ml of Trizol reagent for isolation of the total RNA as de scribed by the manufacturer, All RNA ex tractions were carried out in a chemical hood using Lonafarnib 価格 RNAse free labware. RNA quality and quantity were evaluated by agarose gel electrophoresis and UV spec trometry, Samples were stored at −80 C until used. For reverse transcription reaction, 2 ug of total RNA was reversely transcribed using MMLV RT kits according to the manufacturers protocol, Real time RT PCR A ten fold serial dilution of the cDNA derived from EL 4 cells was prepared in order to make standard curves and determination of PCR efficiency primers for the GM2 GD2 synthase gene and GAPDH housekeeping gene, For performance of real time RT PCR we used a DT 96 PCR machine, and each reaction was performed in a total volume of 20 ul containing 2 ul of cDNA of the test sample or con trol sample with 5xSybrGreen mix prepared according to the manufacturers protocol, Final concentrations of the primer sets and MgCl2 were 10 uM and 3 mM, respectively.

After the denaturation step at 95 C for 5 min, the amplification program was set at 40 cycles each consisting of de naturation at 95 C for 15 s followed by annealing at 58 C for 10s, extension at 72 C for 3 min, followed by detection at the specified acquisition temperature. Melting curve analysis was used for amplicon`s size estimation. Negative controls, samples without reverse transcription or cDNA template were included with every PCR run and were always negative, Relative gene expression was determined as the ratio of the GM2 GD2 synthase gene to the internal reference gene expression based on the Ct values using QGENE software.