Mudanças entre as edições de "What Can be So Interesting Over LY2835219?"
(What Can be So Interesting Over LY2835219?)
Edição atual tal como às 21h28min de 12 de outubro de 2019
The following anti-phosphor-antibodies were used: pPLC��1 (Tyr783), pPLC��2 (Tyr1217), pAkt (Ser473), pPKC (pan), pPKC�� (Thr505), pErk1/2 (Thr202/Tyr204), pJnk (Thr183/Tyr185), p-p38 (Thr180/Tyr182), pI��B�� (Ser32), and pIKK��/�� (Ser176/180) from Cell Signaling Technology. pSNAP-23 (Ser95) was a generous gift from Dr. P. Roche, NCI . Anti-Lipin1 and Lipin2 antibodies were from Santa Cruz Biotechnology. For the loading control, antibodies for total proteins or ��-actin were used. In the stimulation assay, IgE-sensitized BMMCs were resuspended in Tyrode's buffer, and then were left unstimulated or stimulated with DNP-HSA (20 ng/mL) for the various MG-132 times indicated. RNAs extracted using Trizol Reagent (Invitrogen) were reverse transcribed to cDNA by Superscript III and random primers according to the manufacturer's protocol (Invitrogen). qRT-PCR was performed with Mastercycler realplex and SYBR Green master mix (Eppendorf). The expressed levels of target mRNAs were normalized with L32, calculated using the 2?����CT method and presented as arbitrary units (a.u.) of fold change. BMMCs were sensitized with 1 ��g/mL IgE for 6 h, resuspended at 1 �� 107 cells/mL in Tyrode's buffer containing 3 ��g/mL Indo-1 (Invitrogen) and LY2835219 molecular weight 4 mM probencid, further incubated for 30 min at 37��C, and washed twice with Tyrode's buffer. A total of 40 ��l of cells was added into 460 ��l of pre-warmed Tyrode's buffer to determine the calcium response by flow cytometry. After deter-mination of the baseline ratio of FL5 to FL4, cells were stimulated with 10 ��l of 5 ��g/mL DNP-HSA. Calcium flux was displayed as the ratio of FL5 to FL4 fluorescence. Retrovirus was made using pMX-CD63-GFP, kindly provided by Dr. W. Zhang, Duke University according to the method previously described . Isolated BM cells were grown in IMDM-IL3 medium for 3 days. The cells (3 �� 106 cells/mL in 24 wells) were mixed with an equal volume of CD63-GFP retroviral supernatant and polybrene (8 ug/mL), and then centrifuged at 2500 rpm for 90 min at room temperature using a Sorvall Swing Bucket rotor. After spin infection, the CAL-101 cells were further cultured in IMDM-IL3 medium for 4 weeks. The transduced BMMCs were loaded with anti-DNP-IgE and stimulated with DNP-HSA for the indicated times. Cells were immediately fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 followed by incubation with a rabbit anti-lipin1 antibody (Cell Signaling Technology) for 1 h. The bound antibody was labeled with Texas Red-conjugated anti-rabbit secondary antibody (Molecular Probes) for 1 h and then visualized under Leica SP5 confocal microscopy. For statistical analysis, a two-tailed Student's t-test was performed. *p