Gapdh

Proteins (30 μg) were separated by SDS-PAGE, transferred to the PVDF nitrocellulose membrane (Millipore, Boston, MA, USA), blocked with 5% fat-free milk for 2 h at room temperature, and then incubated with primary antibodies in 5% milk overnight at 4 °C. COL3, MMP9 antibodies were all purchased from Wanlei (Wanlei, Shen Yang, China), and GAPDH from Bioworld (Bioworld, Nanjing, China). Rabbit HRP-conjugated secondary antibody (Baoshen, Beijing, China) was added and incubated at room temperature for 2 h. Then proteins were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA), and finally the blots were quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

Antibodies used for this study were directed against phospho-BCR-ABL (210 kDa, 3901S), H3K27me3 (17 kDa, 9733), PARP (89 & 116 kDa), Akt (60 kDa, 9272S), KDM6A (155 kDa, 33510) from CST (Danvers, MA, USA); BCR-ABL (210 kDa, ab187831), TRKA (145 kDa, ab8871), H3K27ac (17 kDa, ab4729) from abcam (MA, USA); Flag (T0003), YY1 (70 kDa, 22156-1-AP), beta actin (42 kDa, 20536-1-AP), c-FOS (60 kDa, 66590-1-lg), CBP (29 kDa, 11149-1-AP) from Proteintech, IL, USA; KDM6A (155 kDa, A302-374A, Bethyl Laboratories, Montgomery, Texas, USA); phospho-AKT (60 kDa, GTX128414, GeneTex, CA, USA); phospho-Erk (44 kDa, 11245), Erk (42/44 kDa, 44204) from SAB, Maryland, USA; GAPDH (36 kDa, AP0063, Bioworld, Nanjing, China); RNA pol II (217 kDa, 39497) from Active Motif, Carlsbad, CA.

Hippocampus tissue (approximately 50 mg) was homogenized in 0.5 mL of radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) for protein extraction and centrifuged at 12,000× g at 4 °C for 15 min. The supernatant was collected and protein concentrations were measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Shanghai, China). Western blotting was performed as previously described [25 (link)]. Briefly, proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotted with antibodies against p-CREB, CREB, BDNF (1:1000; Abcam, San Francisco, CA, USA), and GAPDH (1:40,000; Bioworld, Visalia, CA, USA). The reaction was visualized by enhanced chemiluminescence reagent (Biosharp, Wuhan, China). Bio-Rad imaging system was used to quantify protein levels. All experiments were performed in triplicate.

Cells in culture flasks or plates were lysed in ice‐cold buffer containing protease inhibitor cocktail (Roche). Equal amounts of protein samples were loaded and separated by SDS‐PAGE and transferred to PVDF membranes (Millipore) followed by non‐fat milk or BSA blocking. These blots were incubated at 4°C overnight with primary antibodies against ZEB2 (1:1000 dilution; Proteintech, 14026‐1‐AP), Cleaved‐PARP (1:1000 dilution; Cell Signaling, #9541), N‐cadherin (1:1000 dilution; Cell Signaling, #13116), E‐cadherin (1:1000 dilution; Cell Signaling, #14472), Vimentin (1:1000 dilution; GeneTex, GTX100619), Snail (1:1000 dilution; Cell Signaling, #3879) and GAPDH (1:5000, MB001, Bioworld) followed by incubation with horseradish peroxidase (HRP)‐conjugated secondary antibodies at room temperature. Immunoreactive bands on the blots were detected by ECL chemiluminescence kit (Millipore).

Details of the Western blot protocol are described elsewhere^{36 (link)}. Briefly, total protein was collected using cell lysis reagent containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Protein samples were boiled for 5 min, loaded onto a 10% SDS-PAGE gel for separation, and transferred onto polyvinylidene fluoride (PVDF) membranes at 300 mA for one hour. After blocking with 5% bovine serum albumin (BSA) for 2 hours, the membrane was incubated overnight at 4 °C with primary antibodies against GATA4 (Abcam, USA), dentin sialophosphoprotein (DSPP, Abcam, USA), runt-related transcription factor 2 (RUNX2, Abcam, USA), osterix (OSX, Abcam, USA), osteopontin (OPN, Abcam, USA), osteocalcin (OCN, Abcam, USA), bone morphogenetic protein 4 (BMP4 Abcam, USA), GNAI3 (Abcam, USA) and GAPDH (Bioworld, China). Subsequent to this, the membranes were incubated with secondary antibodies at room temperature for 1 hour, rinsed with Tris Buffered Saline (with Tween-20) three times, and visualized by enhanced chemiluminescence. Semi-quantitative measurements were carried out using Image J software (National Institutes of Health, USA).

Cells were lysed with 0.5% NP40 lysis buffer and western blotting was performed according to the standard protocol. Detection was accomplished with the chemiluminescence ECL plus reagent (Thermo, Grand Island, NY, USA) and the chemiluminescence HRP substrate (Millipore, Billerica, MA, USA), and signals were evaluated by a Tanon 5200Multi scanner (Shanghai, China). Primary antibodies were as follows: HDAC1 (Abcam, Cambridge, UK), HDAC2 (Abcam), HDAC3 (Abcam), cleaved caspase-3 (CST, Danvers, MA, USA), cleaved PARP (CST), PARP (CST), GAPDH (Bioworld, St. Louis Park, MN, USA), K9 acetyl-histone H3 (CST), Bax (CST), P53 (Santa Cruz), PUMA (CST), and HA (CST).