P crkl

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).

Immunoblotting was performed according to a previously described method [27 (link), 28 (link)]. Briefly, after incubation in indicated concentrations of inhibitor, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation lysis buffer. The protein content of lysates was determined with a kit (Bio-Rad, Hercules, CA, USA) and proteins were resolved by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) that was incubated with primary antibodies at the appropriate dilution for 1 h. The blots were then probed with the secondary antibodies and developed with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Little Chalfont, UK). Antibodies against the following proteins were used: phosphorylated (p-)ABL, p-Crk-L, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and p-Aurora, p-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (all from Cell Signaling Technology); Crk-L (Millipore); Aurora A (GeneTex, Irvine, CA, USA); and ABL, c-Myc, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Three independent experiments were performed. Protein band intensity was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Antibodies against USP7, BCR-ABL, Lyn, p-Lyn (Tyr507), STAT5, p-STAT5 (Tyr94), PARP, CRKL, and p-CRKL (Tyr207) were purchased from Cell Signaling Technologies, Inc., (Danvers, MA, USA). The antibodies against USP25 and Caspase-3 were obtained from Proteintech (Wuhan, China). The monoclonal antibodies including anti-Flag, anti-HA, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Medical and Biological Laboratories Co., Ltd (Nagoya, Japan). The anti-Ub antibody was purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). HRP-labeled goat anti-mouse and goat anti-rabbit IgG (H + L) antibodies were purchased from Beyotime Institute of Biotechnology (Nantong, China). ART and cycloheximide (CHX) were purchased from TargetMol (Boston, MA, USA), Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. P5091 and imatinib were purchased Selleck Chemicals Inc. (Houston, TX, USA).

By using the 10% SDS-PAGE gel, 15 μg total protein samples was fractionated by electrophoresis and transferred to a Polyvinylidene fluoride (PVDF) membrane in the Bio-Rad Mini-Protein electro-transfer system (Bio-Rad Laboratories, Inc, CA, USA). Incubation of the membranes in 5% skimmed milk - Tris-buffered saline with Tween 20 (TBST) for 1 h to block non-specific binding, was followed by incubation overnight at 4°C with the antibodies against Bcr-Abl (1 : 1000, Cell Signaling Technology), Oct4 (1 : 1000, Santa Cruz), Sox2 (1 : 1000, Cell Signaling Technology), p-CrkL (1 : 1000, Cell Signaling Technology), Stat5 (1 : 1000, Santa Cruz), p-Stat5 (1 : 1000, Santa Cruz), MDR (1 : 1000, Cell Signaling Technology), CD133 (1 : 1000, Cell Signaling Technology), pAkt (1 : 1000, Santa Cruz), Akt (1 : 1000, Santa Cruz), p-PI3K (1 : 1000, Cell Signaling Technology), PI3K (1 : 1000, Cell Signaling Technology), p-mTOR (1 : 1000, Cell Signaling Technology), CD44 (1 : 1000, Santa Cruz), and β-actin (1 : 500, Santa Cruz). The membranes were then washed and incubated in appropriate horseradish peroxidise (HRP)-conjugated secondary antibodies for 1 h at room temperature, then washed with PBS 3 times. Protein bands were then detected in the enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific Inc, Waltham, MA, USA) and quantification was done using the ImageJ software.