P jak2 tyr1007 1008

Cells were lysed directly in lysis buffer to collect whole cell extracts. Protein samples were separated on polyacrylamide gels, transferred onto nitrocellulose membrane by iblot (Invitrogen) and detected using horseradish peroxidase-conjugated secondary antibodies and chemiluminesce (SantaCruz) exposure of BioMax film (Kodak). The following antibodies were used: antibodies to SOCS3, actin, STAT5 and p-STAT5 (Tyr 694/Tyr 699) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JAK2 and p-JAK2 (Tyr1007/1008) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Acrylamide, bovine serum albumin (BSA), bromophenol blue, 7,12-dimethylbenz[a]anthracene (DMBA), hydroxyurea, 2-mercaptoethanol, sodium dodecyl sulphate (SDS) N,N,N′,N′ - tetramethylene diamine (TEMED) and Trizol were purchased from Sigma Chemical Company, St. Louis, MO, USA. Astaxanthin was procured from Bio-Real, Sweden. DMEM-F12 medium, antibiotic solution consisting of penicillin and streptomycin and Alamar blue were from HiMedia Labs, Mumbai, India. Fetal bovine serum of South American origin was from GIBCO, Invitrogen, NY, USA. Power SYBR Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for IL-6, GAPDH, Cyclin D1, PCNA, p21, MMP-2, MMP-9, TIMP-2, RECK, VEGF, VEGFR2, HIF1α, were purchased from Santa Cruz Biotechnology, USA. pJAK-2^tyr1007/1008, JAK-2, pSTAT-3^tyr705, STAT-3 and histone (H2B) antibodies and BrdU, STAT-3^tyr705, total Cyclin D1 and pVEGFR2^tyr1175 ELISA kits were from Cell Signaling Technology, USA. CD-34 antibody was purchased from Novocastra, Germany. Matrigel was from BD Biosciences, USA. All other reagents used were of analytical grade.

The Chinese medicinal herb compound library was a collection of 182 natural compounds isolated from 69 traditional Chinese medicinal herbs (unpublished results). The compounds were dissolved in DMSO at a concentration of 10 mM. The final concentration of a compound in the screening assay was 10 μM. The library screening was performed using a cellular luciferase gene reporter assay as described below. EB (>98% purity) was purchased from Shanghai Boylechem Co.ltD. Stattic was purchased from Selleck Chemicals. Sodium orthovanadate and DAPI (Diamidino-phenyl-indole) were purchased from SIGMA. Antibodies against STAT3, p-STAT3 Tyr705, p-STAT3 Ser727, STAT1, p-STAT1 Tyr701, STAT5, p-STAT5 Tyr694, JAK2, p-JAK2 Tyr1007/1008, JAK1, p-JAK1 Tyr1022/1023, Tyk2, p-Tyk2 Tyr1054/1055, PARP and actin were purchased from CST (Cell Signaling Technology). Antibodies against tubulin were purchased from Santa Cruz Biotechnology. Secondary HRP-conjugated antibody was purchased from MultiSciences Biotech. Alexa Fluor 488 donkey anti-mouse IgG antibody was purchased from Invitrogen.

K562 CD34⁺ cells and primary CML CD34⁺ cells(2.5 × 10⁵ cells/well in 6-well plates) were exposed to various stimulating conditions for the indicated times. The lysated preparations were carried out by Western blot as previously mentioned. Antibodies for Western blot were used in our research: p-JAK2^Tyr1007/1008, p-STAT5^Tyr694, JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA). SHP-1 and cyclin D2 were ordered from Abcam(Cambridge, MA, USA).

Antibodies against p-Tyr701-STAT1 (product #9167), p-Tyr690-STAT2 (product #88410), p-Tyr705-STAT3 (product #9145), p-Tyr1022/1023-JAK1 (product #3331), p-Tyr1007/1008-JAK2 (product #3776), and p-Tyr1054/1055-Tyk2 (product #9321) were purchased from Cell Signaling Technology, USA. Recombinant human STAT3 was obtained from Abcam, UK (product #ab268982). Protease inhibitors and phosphatase inhibitors were obtained from Merck Life Science, USA. Marine natural products library was built in house in the Laboratory for Marine Resources Biology at Ocean University of China.
Six-week old female NRMI congenitally athymic nude (nu/nu) mice (SPF degree, 17–20 g weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Antibodies against p-Tyr1022/1023-JAK1 (#3331), p-Tyr1007/1008-JAK2 (#3776), p-Tyr980/981-JAK3 (#5031), p-Tyr1054/1055-TYK2 (#9321), p-Tyr701-STAT1 (#9167), p-Tyr705-STAT3 (#9145), p-Tyr694-STAT5 (#9359), JAK1 (#3332S), JAK2 (#3230), JAK3 (#8863), TYK2 (#9312), STAT1 (#14994), STAT3 (#12640), STAT5 (#25656), c-Myc (#13987), cyclin D1 (#AF0931), Bcl-xL (#2764), p-Ser536-NF-κB (#3033), p-Thr308-AKT (#9275), p-Ser9-GSK-3β (#5558), p-Thr183/Tyr185-SAPK/JNK (#4668), NF-κB (#8242), AKT (#4691), GSK-3β (#9832), and SAPK/JNK (#9252) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protease inhibitor (B14001) and phosphatase inhibitor (B15001) were purchased from Bimake (Houston, TX, USA). Recombinant human STAT3 protein (ab268982) was obtained from Abcam (Cambridge, Britain). Recombinant human interleukin-6 (IL-6) protein (200-06) was purchased from PeproTech (Rocky Hill, CT, USA). Nitrocellulose membranes and chemiluminescent horseradish peroxidase (HRP) substrate were obtained from Millipore (Billerica, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA). DAB color solution, hematoxylin solution, and eosin staining solution were purchased from Servicebio (Wuhan, China).