Cryptotanshinone (CPT) was purchased from Shanghai Yuanye Biotechnology Co., Ltd, with a purity of 98% (#B21586, Shanghai, China). High-performance liquid chromatography (HPLC) analysis was applied to further confirm the purity of CPT (98%). Imatinib (Genike) was obtained from Nanjing Chia Tai Tianqing Pharmaceutical Co., Ltd. The second-generation tyrosine kinase inhibitors nilotinib was purchased from Aladdin Chemistry Co., Ltd (#N126111, Shanghai, China). Dasatinib was obtained from Shanghai Yuanye Biotechnology Co., Ltd (#S45672, Shanghai, China). Antibodies used in this study were purchased from Cell Signalling Technology (MA, USA), including cleaved caspase-3 (#9664), cleaved PARP (#5625), p-STAT3 (#9145), Bcr-Abl (#3908), p-Bcr-Abl (#3009), p-Src (#2101) and β-Actin (#3700). Antibody for cleaved caspase-9 (Asp353) (#AF5240) was obtained from Affinity Biosciences (Cincinnati, OH, USA), for eIF4E (#610270) was obtained from BD Biosciences (San Diego, CA, USA), and p-eIF4E (ab76256) was purchased from Abcam. The rabbit polyclonal antibody against Ki-67 (PB9026) and mouse monoclonal antibody against PCNA (BM0104) was obtained from Boster Biological Technology Co., Ltd.
P bcr abl
Manufactured by Cell Signaling Technology
Sourced in United States
P-Bcr-Abl is a phospho-specific antibody that detects the phosphorylation of the Bcr-Abl fusion protein. Bcr-Abl is a constitutively active tyrosine kinase that plays a central role in the development of chronic myelogenous leukemia (CML).
Automatically generated - may contain errors
Lab products found in correlation
Bcr abl, by Cell Signaling Technology (7 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Cleaved parp, by Cell Signaling Technology (3 mentions)
P stat5, by Cell Signaling Technology (3 mentions)
P crkl, by Cell Signaling Technology (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Stat5, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
P src, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
Survivin, by Cell Signaling Technology (2 mentions)
Cryptotanshinone, by Yuanye Bio-Technology (1 mentions)
P eif4e, by Abcam (1 mentions)
Dasatinib, by Yuanye Bio-Technology (1 mentions)
Pb9026, by Boster Bio (1 mentions)
Eif4e, by BD (1 mentions)
7 protocols using p bcr abl
1
Evaluation of Cryptotanshinone in Leukemia
2
Investigating Imatinib Resistance in CML
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The human CML cell line KBM5 (harboring BCR-ABL) and its IM-resistant sub-line with T315I mutation (KBM5-T315I cells) were cultured at 37 °C with 5% CO2 in Iscove’s Modified Dulbecco’s Media (IMDM, from Gibco, USA) supplemented with 10% fetal bovine serum (FBS, from Hyclone, USA) as we described previously [9 (link)]. KBM5-T315I cells were routinely maintained in IMDM medium with IM (1.0 μM), which was removed 2–3 days before KBM5-T315I cells were used for experiments. IM was purchased from Selleckchem (Houston, TX, USA) and 2-DG was obtained from Cayman Chemical (Ann Arbor, MI, USA). Annexin V-FITC/PI apoptosis kit was from BD Biosciences (San Jose, CA, USA). Seahorse base medium, oligomycin, Carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and rotenone/antimycin A were obtained from Seahorse Bioscience (North Billerica, MA, USA). Antibodies against AMPK(#5832), p-AMPK(#2537), Bcr/Abl(#3908), p-Bcr/Abl (#3901),beclin-1 (#4122), intact and cleaved caspase-8 (#9748, #9746), cleaved PARP (#5625), Glut1 (#12939), Glut4 (#2213), HK I (#2024), HK II (#2106), LC3A (#4599), mTOR (#4517), p-mTOR (#5536), p-p70S6K (Thr389) (#97596), p70S6K (#34475), p-CrkL (#3181), p-SRC (#2101), p-STAT5 (#4322), β-actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to total OXPHOS cocktail (#110411) was purchased from Abcam (Cambridge, MA, USA).
3
Immunoblotting of Apoptosis Markers
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We used the following antibodies for immunoblotting: BCR-ABL (#2862), p-BCR-ABL (#2861), BIM (#2819), CASPASE 3 (#9662), STAT5 (#9310), p-STAT5 (#9359), PARP (#9542) (all from Cell Signaling Technology, USA) and beta-ACTIN (#AC-15, Sigma, USA). The antibody dilutions used were 1 in 1,000, except for PARP (1 in 2,000), BIM (1 in 500) and β-ACTIN (1 in 5,000). HRP-conjugated secondary antibodies were specific to rabbit (Sigma, USA) or mouse IgG (Santa Cruz biotechnology, USA). The protein bands on the membrane were visualized using the Western Lightning chemiluminescence reagent (PerkinElmer, USA).
4
Apoptosis and Autophagy Signaling Pathway
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Caspase-3 (#9665; Cell Signaling Technology, Danvers, MA, USA), Bcl2 (#2870; Cell Signaling Technology), Bax (#2772; Cell Signaling Technology), P62 (#7695; Cell Signaling Technology), Akt (#9272; Cell Signaling Technology), p-Akt (#4060; Cell Signaling), mTOR (#2983; Cell Signaling Technology), p-mTOR (#5536; Cell Signaling Technology), LC3 (L7543; Sigma-Aldrich, St Louis, MO, USA), Cav-1 (#3267; Cell Signaling Technology), Bcr-Abl (#2862; Cell Signaling Technology), p-Bcr-Abl (#3901; Cell Signaling Technology), CrkL (#3182; Cell Signaling Technology), p-CrkL(Tyr207) (#3181; Cell Signaling Technology), and β-actin (TA-09; ZSGB-BIO, Beijing, China) were used in the present study. Horseradish peroxidase–labeled secondary antibodies anti-mouse IgG (ZB-5305; ZSGB-BIO) and anti-rabbit IgG (ZB-2301; ZSGB-BIO) and fluorescence-labeled secondary antibodies goat anti-mouse IgG (ZF-0512; ZSGB-BIO) and anti-rabbit IgG (ZF-0511; ZSGB-BIO) were also used.
5
Protein Expression Analysis of HS-543 and Imatinib
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After the cells were treated with various concentrations of either HS-543 or Imatinib and incubated at 37°C for various times, they were collected and washed with cold phosphate-buffered saline (PBS). Then, the cells were lysed with a RIPA buffer (BIOSESANG, Korea) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX). The proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred onto the nitrocellulose membranes. The blots were immunostained with the appropriate primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase. Antibody binding was detected with an enhanced chemiluminescence reagent (Bio-Rad. Hercules, CA). Primary monoclonal antibodies against the following factors were used; p-Bcr-Abl (Tyr177), p-Crkl (Tyr207), p-Stat5 (Tyr694), Bcr-Abl, Crkl, Stat5, cleaved PARP, cleaved caspase-3, cytochrome c, survivin, Mcl-1, Bax and β-actin (Cell Signaling Technology, Danvers, MA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX).
6
Molecular Signaling Pathway Analysis
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The small molecules b‐AP15, ZVAD, imatinib and bortezomib were purchased from Selleck Chemicals and dissolved in DMSO and stored at –20°C. The final dose of DMSO limited to 0.3% in all experiments. The antibodies as follows were obtained from Cell Signaling Technology (Danvers, MA): PARP (#9532S), p27 (#3688S), USP14 (#11931S), Caspase‐3 (#9662S), Caspase‐8 (#9746S), Caspase‐9 (#9508S), XIAP (#2042S), Bax (#5023), Bcl‐2 (15071S), Survivin (#2808S), AIF (#4642S), Cytochrome C (#4272S), PERK (#5683S), ATF4 (#11815S), CHOP (#2895), BCR‐ABL (#2862S), p‐BCR‐ABL (#2861S), STAT5 (#9363S), p‐AKT (#2965), AKT (#4685), CRKL (#3182S) and p‐CRKL (#3181S). The antibodies as follows were obtained from Proteintech (Rosemont, USA): Mcl‐1 (#16225‐1‐Ap) and BCL‐xl (#66020‐I‐Ig). The antibodies as follows are purchased from Abcam (Cambridge, MA): Cleaved Caspase 9 (#AB3629) and Cleaved Caspase 3 (#AB2302). The antibody of ubiquitin was purchased from Santa Cruz Biotechnology. p‐STAT5 (#50095), anti‐mouse (12‐349) and anti‐rabbit (AP132P) antibodies were obtained from Merk Millipore (Darmstadt, Germany). All the above antibodies were used at a dilution of 1:1000.
7
Panobinostat and Sodium Butyrate Induce Apoptosis in CML Cell Lines
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Human CML cell line K562 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). K562/ADR, an adriamycin–resistant cell line, and K562/G, an imatinib–resistant cell line, were kindly provided by Dr. Wei He (Institute of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, China). All cells were maintained in RPMI‐1640 medium (Hyclone Laboratories) supplemented with 10% defined FBS (Hyclone Laboratories). Cells were cultured at 37°C in 5% CO2. Panobinostat (LBH589) was purchased from Selleck Chemicals (Houston, TX). Sodium butyrate (NaBu) was purchased from Sigma‐Aldrich (St. Louis, MO). The primary antibodies against β‐ACTIN, GAPDH, caspase‐3, caspase‐6, caspase‐8, caspase‐9, PARP, BIP, BCL‐XL, MCL‐1, BAX, BAK, BAD, XIAP, survivin, ABCG2, MDR1, BCR/ABL, p‐BCR/ABL, AKT, p‐AKT, mTOR, p‐mTORC1, 4EBP1, p‐4EBP1, eIF4E, p‐eIF4E and c‐MYC were purchased from Cell Signaling Technology (Danvers, MA), the antibody against caspase‐7 was purchased from Abcam (Cambridge, UK).
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