Cells were toward put on ice for 10 min and washed twice in cold PBS. Cell pellets were then resuspended in 250 ml of buffer A and incubated on ice for 10 min. After centrifugation at 15,000 g for 10 min at 4 C, supernatant were kept to obtain the cytosolic frac tions. The cytosolic fraction was mixed with an equal volume of 2�� RIPA buffer. Ribonuclease assay The RNase activity of the recombinant proteins against a yeast tRNA substrate was measured in 20 mM Tris HCl buffer at 37 C. Purified RNase was added into 50 ul of the Tris buffer with 120 ug of tRNA. The reaction was stopped by addi tion of 200 ul 0. 7% perchloric acid with 0. 1% uranyl acetate and incubated on ice for 30 min. The insoluble tRNA was removed by centrifugation at 14,000 g for 15 min at 4 C.
The amount of solubilized tRNA was deter mined by Inhibitors,Modulators,Libraries UV absorbance at 260 nm. The catalytic activ ity of the RNase was determined Inhibitors,Modulators,Libraries as the nanogram of RNA digested per second per nanomol of RNase used. Statistical analysis Results were described as mean standard deviation. All statistical analysis was conducted by the statistical package SPSS13. 0. The differences were investigated using Students t test and one way analysis of variance. Values of P are considered to be statistically significant P 0. 05, P Inhibitors,Modulators,Libraries 0. 01. P 0. 001. List of abbreviations Ad12SV40 adenovirus 12 SV40 virus hybrid. AECs air way epithelium cells. ECP eosinophil cationic protein. rECP recombinant ECP. mECP H15A K38I H128A mutant rECP. EDN eosinophil derived neurotoxin. ER endoplasmic reticulum. FACS fluorescent activated cell sorting.
GRP78 78 kDa glucose regulated protein. HS heparan sulfate. HSPG heparan sulfate proteoglycan. MMP mitochondrial membrane potential. MTT 3 2,5 diphenyltertrazolium bromide. ONC onconase. PARP Inhibitors,Modulators,Libraries poly polymerase. pI isoelectric point. PI propidium iodide. PNK Protei nase K. RNase ribonuclease. STS staurosporin. TG thapsigargin. TNF a tumor necrosis factor alpha. TNFR1 TNF receptor 1. TRADD TNFR associated death domain. TRAIL TNF related apoptosis inducing ligand. Z LE HD FMK benzyloxycarbonyl Leu Glu His Asp fluoromethylketone Z IETD FMK Benzyloxycarbonyl Ile Glu Thr Asp fluoro methylketone. Z VAD FMK Benzyloxycarbonyl Inhibitors,Modulators,Libraries Val Ala Asp fluoromethyl ketone. Z ATAD FMK benzylox ycarbonyl Ala Thr Ala Asp fluoro methylketone.
Background HS 1 associated protein X 1, Hax 1, is a 35 kDa pro tein with two Bcl 2 homology domains that was identified in a yeast two hybrid screen where it was found to interact with HS 1, a Src kinase substrate. Hax 1 is ubiquitously expressed in most tissues and is reported to be localized in mitochondria as well as the endoplasmic reticulum several and nuclear membrane. Mutations identified in the human HAX 1 gene have been shown to cause neutropenia and neurodeve lopmental abnormalities. Knockout HAX 1 mice show increased apoptosis of neurons and postnatal le thality.