To detect the tyrosine phosphorylated EpoR, phosphotyrosyl proteins from 500g of total cell protein have been immunoprecipitated with mAb 4G10 in IP buffer Tween20, 10g ml aprotinin, 1 mM PMSF, 5g ml antipain, 0. 5g ml leupeptin, 0. 7g ml pepstatin A, 1 Comprehensive inhibi tors, 3 mM DTT, 1 mM NaF, 1 mM Na3VO4 overnight in the cold. Soon after SDS Web page and membrane transfer, EpoR was detected with polyclonal anti EpoR and ECL. PI3 kinase assay 500g of total cell protein extract had been immunoprecipi tated in NP 40 lysis buffer with 4g anti phosphotyrosine mAb or with 4g PI3K p110 polyclonal anti physique, Santa Cruz respectively. Immu noprecipitates had been washed 3 instances with NP 40 lysis buffer, twice with 50 mM TrisHCl pH 7. 5 plus 500 mM lithium chloride, as soon as with 20 mM TrisHCl pH 7.
five with one hundred mM NaCl and 1 mM EDTA and after with 20 mM HEPES pH 7. five. 10g of lyophilised phosphatidyli nositol had been selleck then mixed with ten l of lipid kinase buffer and sonicated for 6 5 sec at 30% energy output with an MS72 sonotrode, chilling PI on ice amongst sonications. Sonicated PI was then mixed with 30 l LKB and added towards the immunoprecipitates. After 5 min on ice, 10 l LKB with 10 Ci 32P ATP and 2 mM magnesium chlo ride were added and samples incubated at 37 C for 15 min. The reaction was stopped with 200 l of 1 N hydrochloric acid. Just after extraction of the samples with 200 l of methanol chloroform, 50 l on the organic phase have been made use of for thin layer chromatography on LK6D plates. Running buffer was methanol chlo roform H2O NH4OH. TLC plates were analyzed on a Storm860 phosphoimager with ImageQuant 5. 2.
To confirm equal PI3K precipitation independent of cell stimulation, parallel samples of immunoprecipitates from protein extracts ana lyzed in lipid kinase assay had been separated by SDS Web page and blots probed with anti p110 mAb followed by ECL. Raf MEK coupled kinase assay The coupled kinase assay was accomplished basically as described with slight modifications. 500g of total cell protein extract were epigallocatechin immunoprecipitated with 4g anti c Raf1 or with 4g anti B Raf in buffer A TritonX 100, 10% glycerol. Immunoprecipitates had been washed three times with buffer A and twice with buffer B. 0. 2g acti vatable GST MEK1 with each other with five l buffer C and 10 l buffer B were added to the washed immunoprecipitates, samples have been mixed and incubated at 30 C for 20 min. The reaction was then disrupted by transferring samples into ice and adding 100 l buffer D. 25 l of this reaction mix was subsequently incubated with 20g kinase inactive GST Erk1K63M, two l buffer E and five Ci 32P ATP at 30 C for 15 min. The reaction was stopped by adding SDS Page sample buffer. Just after SDS Page, Raf activity was determined by detecting GST Erk1K63M phosphorylation with Image Quant five.