Anti protein kinase b akt

Immunoblot assay was performed as previously described [66 (link)]. Conditioned medium was collected and concentrated by Amicon Ultra-4 Centrifugal Filters Ultracel-30K (Merck Millipore). The blots were stained with InstantBlue (Expedeon Inc. San Diego, CA, USA). Primary antibodies were used as follows: anti-IGFBP3 (MAB305, R&D Systems), anti-integrin β1 (Santa Cruz, Dallas, TX, USA), anti-α-tubulin (MS-581-P0, Thermo Scientific), anti-focal adhesion kinase (FAK, sc-557, Santa Cruz), anti-phosphorylated FAK (611806, BD), anti-Src (#2109, Cell Signaling, Beverly, MA, USA), anti-phosphorylated Src (05–677, Millipore), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB, sc-8008, Santa Cruz), anti-phosphorylated NF-kB (#3033, Cell Signaling), anti-protein kinase B/AKT (#9272, Cell Signaling), anti-phosphorylated AKT (#9271, Cell Signaling), anti-ERK (sc-94, Santa Cruz). anti-phospho-ERK (sc-7383, Santa Cruz), anti-integrin-linked kinase (ILK, GTX101691, GeneTex, Irvine, CA, USA), MMP1 (RB1536P0, Thermo Scientific) and MMP10 (AP6194a, Abgent, San Diego, CA, USA).

Anti-extracellular signal-regulated kinase (ERK), anti-phospho-ERK, anti-p38 mitogen-activated protein kinase (MAPK), anti-phospho-p38 MAPK, anti-Janus kinase (JNK), anti-phospho-JNK, anti-protein kinase B (AKT), and anti-phospho-AKT antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Polyclonal anti-cyclin D1, anti-cyclin E, anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-p53, anti-p21WAF1, anti-p27KIP1, and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Moreover, a Ki-67-specific antibody was obtained from Invitrogen (Waltham, MA, USA). Moreover, a nuclear extract kit and electrophoretic mobility shift assay (EMSA) gel shift kit were purchased from Panomics (Fremont, CA, USA). SP600125 were purchased from Calbiochem (San Diego, CA, USA).

Antithrombin, rapamycin, A6730, Evans blue, and triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-beclin-1 (1:1000), anti-GAPDH (1:5000), anti-protein kinase B (Akt; 1:1000), anti-phosphorylated (p)-Akt (Ser473,1:1000), anti-autophagy protein microtubule-associated protein 1 light chain-3B (LC3B; 1:1000), anti-B-cell lymphoma 2 (Bcl-2; 1:1000), anti-Bcl-2 associated x (Bax; 1:1000), and anti-p62 (1:1000) antibodies were obtained from either Cell Signaling Technology (Danvers, MA) or Abcam (Cambridge, UK). Phenylmethane sulphonyl fluoride (PMSF), the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit, and the bicinchoninic acid protein quantification kit were obtained from Beyotime (Shanghai, China). The creatine kinase (CK) assay kit was purchased from the Jiancheng Bioengineering Institute (Nanjing, China). Goat anti-rabbit secondary antibodies were purchased from Biosharp (Guangzhou, China).

Proteins were extracted with lysis buffer and western blot analysis was performed as previously reported (Forte et al. 2022 ). The following primary antibodies were used: anti-SIRT3 (5490S, Cell Signaling Technology, Danvers, MA, US), anti-protein kinase B (AKT) (9712, Cell Signaling Technology), anti-phosphoAKT (Ser-473, 12694, Cell Signaling Technology), anti-5’ AMP-activated protein kinase (AMPK) (2603, Cell Signaling Technology), anti-phosphoAMPK (Thr172, 2535, Cell Signaling Technology), anti-MHC (ab50967, abcam), anti-ANP (ab225844, abcam), anti-GAPDH (97,166, Cell Signaling Technology), anti-Vinculin (sc-73,614, Santa Cruz Biotechnology, Dallas, TX, US), anti-β-ACTIN (sc69879, Santa Cruz Biotechnology), anti-Manganese Superoxide Dismutase (MnSOD) (NBP2-20535, Novus Biological, Centennial, CO, US). Secondary antibodies were anti-rabbit (AP132P, Millipore, Burlington, MA, US) and anti-mouse (Millipore, 401,215).

After, the specified treatments, ice-chilled PBS was employed to wash the PAFs twice. Afterwards, 0.15 ml cell lysis buffer (Cat No P0013, Beyotime Institute of Biotechnology, China) enriched with protease inhibitor was employed to lyse the cells for 15 minutes. Next, the BCA kit (Cat No, Beyotime Institute of Biotechnology, China) was employed to quantify the proteins. Subsequently, 30 μg/lane proteins were fractionated on a 10% SDS-PAGE gels. The fractionated proteins were blotted onto PVDF membranes (300 mA for one hour). The membranes were inoculated with the primary antibodies and incubated at 4℃ overnight. Thereafter, the membranes were inoculated with the secondary peroxidase-labelled antibody for one hour. The protein bands were detected via enhanced chemiluminescence and exposure to ECL Hyperfilm (GE Healthcare). NIH Image 1.63 software was employed to quantify the densitometry of bands. Beta-actin served as the normalization standard. The primary antibodies used included anti-phospho-PDK1 (1:1,000), anti-protein kinase B (AKT) (1:1,000) and anti-phospho-AKT (Ser 473; 1:1,000) (All from Cell Signaling Technology, Danvers, MA, USA). Antibodies against β-actin (1:1,000) and HIF-1 α (1:2,500) were from Genetex, Inc. (Genetex, CA, USA).