Anti pten

PE conjugated anti-CD34 and anti-CD45 primary antibodies were from BioLegend (USA). The collagenase type II was from Sigma (St. Louis, MO, USA). Ham’s/F-12 medium and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Trypsin was purchased from Gibco (Billings, MT, USA). Penicillin and streptomycin were from Sorlabio (Beijing, China). Fibroblast growth factor was from Peprotech (Rocky Hill, NJ, USA). Leukocyte inhibitory factor was product of Gibco. Rabbit anti-rat c-kit⁺ primary antibody was supplied by Biorbyt (Cambridge, UK). M-280 beads conjugated with sheep anti-rabbit secondary antibody were from Dynal Biotech (Hovik, Norway). miR-21 mimics and the negative control scramble were synthesized by RIBOBIO (Guangzhou, China). Lipofectamine 2,000 was from Invitrogen (Carlsbad, CA, USA). Primers, miRNA reverse transcript kit and qRT-PCR kit were from Sangon Biotech (Shanghai, China). Anti-β-Actin, anti-PTEN, anti-BrdU, anti-P-Akt, anti-Akt primary antibody, and other secondary antibodies were obtained from Boster bio (Wuhan, China). EdU (5-ethynyl-29-deoxyuridine) cell proliferation detecting kit was from RIBOBIO (China). Cell cycle detecting kit was from KeyGEN (Nanjing, China). LY294002 (PI3K inhibitor) was from Beyotime (Jiangsu, China). Phen (PTEN inhibitor) was product of Merck (Darmstadt, Germany). The unlisted reagents were of analytical grade.

PC12 cells were placed in ice-cold lysis buffer for 30 min (Beyotime). The supernatant was collected via centrifugation at 1,120 × g for 20 min at 4°C. Protein concentration was calculated using a BCA kit (Pierce, USA). Then, the proteins were separated using 10% SDS-PAGE and transferred onto PVDF membranes, which were blocked with 5% nonfat milk. This was followed by overnight incubation at 4°C with the following primary antibodies (1:1000): anti-caspase3, anti-bcl-2, anti-bax, anti-PTEN (Boster Biological Technology, Wuhan, China). The proteins were then incubated with goat–anti-rabbit antibody (Abcam, Shanghai) and treated with BeyoECL Plus (Beyotime, Shanghai). Protein bands were visualized using a ECL kit (KeyGEN BioTECH, Nanjing, China).

PE conjugated anti-CD34 and anti-CD45 primary antibodies were from BioLegend (USA). Collagenase type II was from Sigma (USA). Ham's/F-12 medium and fetal bovine serum (FBS) were purchased from HyClone (USA). Trypsin was purchased from Gibco (USA). Penicillin and streptomycin were from Solarbio (China). Fibroblast growth factor was from Peprotech (USA). Leukocyte inhibitory factor was a product of Gibco (USA). Rabbit anti-rat c-kit⁺ primary antibody was supplied by Biorbyt (UK). M-280 beads conjugated with sheep anti-rabbit secondary antibody were from Dynal Biotech (Norway). miR-21 mimics, miR-21 inhibitor, and their scrambles were synthesized by RIBOBIO (China). Lipofectamine 2000 was from Invitrogen (USA). Primers, miRNA reverse transcription kit, and qRT-PCR kit were from Sangon Biotech (China). Anti-β-actin, anti-Bcl-2, anti-Bax, anti-caspase-3, anti-PTEN, anti-p-Akt, and anti-Akt primary antibody and other secondary antibodies were obtained from Boster (China). Annexin V-FITC apoptosis detecting kit was from Solarbio (China). LY294002 (PI3K inhibitor) was from Beyotime Technology (China). Phen (bpV, PTEN inhibitor) was a product of Merck (Germany). The unlisted reagents were of analytical grade.