Polyvinyl difluoride pvdf membrane

DMEM, propidium iodide (PI), acridine orange, monodansyl cadaverine, 3-(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT), bafilomycin, chloroquinone (CQ), sodium fluoride, sodium orthovanadate, fetal bovine serum were purchased from Sigma-Aldrich, Missouri, USA. Akt inhibitor IV and VIII, antibodies of caspase-3, cyclin A, cyclin B, cdc2, PARP-1, β-Actin and Akt siRNA were from Santa Cruz Biotechnology, Texas, USA. Antibodies to cdc25, CDK-1, cyclin E were purchased from Piercenet, Illinois, USA. All other antibodies and chemicals were purchased from Cell signaling technology, Massachusetts, USA. Electrophoresis reagents, reagents for protein estimation and protein molecular weight markers were from Bio-Rad Laboratories, California, USA. Polyvinyldifluoride (PVDF) membrane was purchased from Millipore, Massachusetts, USA. K-LISA mTOR activity kit were purchased from Calbiochem, San Diego, USA.

hTCEpi cells or HCECs were lysed directly in 6-well culture plates using radioimmunoprecipitation (RIPA) buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher, Rockford, IL) on ice for 10 minutes, then centrifuged for 5 minutes at 12,000 rpm at 4 °C in a microcentrifuge (BioRad, Hercules, CA). Protein concentration was determined using a Qubit 3.0 Fluorometer (Thermo Fisher, Rockford, IL). Supernatants were removed and boiled for 5 minutes in 2× sample buffer pH 6.8 containing 65.8 mM Tris-HCL, 26.3% (w/v) glycerol, 2.1% SDS, 5.0% β-mercaptoethanol and 0.01% bromophenol blue (Bio-rad, Hercules, CA). Samples were resolved on a 4–15% precast linear gradient polyacrylamide gel (Bio-rad, Hercules, CA) and transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, Temecula, CA). Membranes were blocked in 5% non-fat milk (Bio-rad, Hercules, CA) for 1 hour at room temperature and incubated in primary antibody overnight at 4 °C. Following a 1 hour incubation with an anti-mouse or anti-rabbit secondary antibody (Santa Cruz, CA), protein was visualized using ECL Prime Detection Reagent (Amersham Biosciences, Piscataway, NJ) and imaged on an Amersham Imager 600 (Amersham Biosciences, Piscataway, NJ). For immunoblots of whole cell lysates, β-actin or GAPDH were used as loading controls.

For western blot detection of P53, P16, and P21, proteins were solubilized in 8 M Urea lysis buffer (8 M Urea, 50 mM Tris-HCl, 0.1 M β-Mercaptoethanol, 1 mM DTT, 100 μg/ml phenylmethanesulfonylfluoride). Proteins (15 μg) were separated by 8% (P53) or 14% (P21, P16) SDS-PAGE, blotted onto polyvinyl difluoride (PVDF) membrane (Millipore, Corporation, Billerica, MA, USA), and revealed using the anti-P53 DO-7 mAb (1/1000; Dako, Glustrup, Denmark), the anti-P21 EA10 mAb (0.5 μg/ml; Abcam ab16767, Cambridge, UK), or the anti-human-P16 mAb (1/1 000, BD Biosciences, Le Pont de Claix, France). Membranes were re probed using the anti-GAPDH mAb (1/2000; Abcam 9484, Cambridge, UK) or the anti-β-Tubulin TUB2.1 mAb (1/5 000, Sigma Aldrich, St Louis, MO) for loading/transfer controls. Membranes were then analysed using the Luminata crescendo western HRP substrate (Millipore Corporation, Billerica, MA, USA). Quantification of P53 was performed using the ImageJ software. Quantification of P21 and P16 was performed using the Fusion-Capt software (Vilber Lourmat, Eberhardzell, Germany). For detection of SHH, 1μg of concentrated culture supernatants were subjected to 10% SDS-PAGE, blotted, and revealed using the N-SHH 167Ab polyclonal antibody [26 (link)]. Quantification of N-SHH was performed using GeneGnome device and GeneTools software (Syngene, Synoptics Ltd, Cambridge, UK).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, 2 mM l-glutamine, lipofectamine 2000, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture dishes, 6-well, and 12-well plates were purchased from Greiner Bio-One (Frickenhausen, Germany). Polyvinyldifluoride (PVDF) membrane and an Immobilon Western Chemiluminescent HRP Substrate detection system were purchased from Millipore (Billerica, MA, USA). All of the primary antibodies specific for IL-6 (sc-28343), CXCR2 (sc-32780), c-Raf (sc-7267), MEK (sc-6250), ERK (sc-514302), JNK (sc-7345), p38 (sc-81621), c-Jun (sc-166540), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit antibodies specific for phosphorylated forms of c-Raf (#9427), MEK (#3958), ERK (#4376), JNK (#9255), p38 (#9216), and c-Jun (#2361) were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). All inhibitors against signal pathway components were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human CXCL1 was obtained from PeproTech (Rocky Hill, NJ, USA). All small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Random hexanucleotides, dNTPs, enhanced chemiluminescence (ECL) reagents, Taq polymerase, RNase inhibitor, and reverse-transcriptase enzymes were obtained from the New England Biolabs (USA); DTNB, acetyl thiocholine iodide, TRI reagent, monoclonal anti-β-actin-peroxidase (A3854) and rabbit polyclonal GFAP antibody (G4546), and mini protease inhibitor cocktail were purchased from Sigma-Aldrich (USA). Mouse monoclonal Arc antibody (sc-17839) and goat polyclonal BDNF antibody (sc-33904) were purchased from Santa Cruz Biotechnology, USA. Horse radish peroxidase conjugated secondary antibodies were purchased from Bangalore Genei, India, and polyvinyl difluoride (PVDF) membrane was procured from Millipore (Germany). CDRI-08 extract of B. monniera was generously provided by Lumen Marketing Company, Chennai. All other biochemicals were purchased from Merck (Germany).

DNA marker and 2x Tag Master Mix were purchased from Beijing CoWin Biotech (Beijing, China). Polyvinyl difluoride (PVDF) membrane was obtained from Millipore (Billerica, MA, USA). PageRuler Prestained Protein Ladder was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against TRAF1 and Bcl-xL were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Millipore. Anti–4-1BB antibody was obtained from Abcam (Cambridge, MA, USA). Goat anti-rabbit and anti-mouse immunoglobulin G (IgG) horseradish peroxidase- (HRP-) conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise stated, all other reagents were provided by the Central South University Cancer Research Institute, Changsha, China.