Hrp conjugated antibodies

Primary antibodies against Furin (70393), Vimentin (5741), N-cadherin (14215), and β-Actin (4970) were purchased from Cell Signalling Technology (Danvers, MA, USA). Those against β-Catenin (ab32572), hsc70 (ab51052), and GAPDH (ab8245) were from Abcam (Cambridge, UK). Other primary antibodies used were: E-cadherin (80182) from BD Biosciences (Bedford, MA, USA) and α-SMA (14-9760-82) from Invitrogen™ (Waltham, MA, USA). All the HRP-conjugated antibodies were from DAKO (Nowy Sącz, Poland) and for immunofluorescence detection Alexa Fluor 488 anti-rabbit IgG (Invitrogen™) and Cy3 anti-mouse (Sigma-Aldrich, St. Louis, MO, USA) were used. For the ChIP assay, antibodies against RARα (sc-515796) and non-specific IgG (sc-2025) were used, both from Santa Cruz Biotechnology (Dallas, TX, USA).

Lysis buffer (12.5 ml Tris HCL, 2g SDS, 10 ml Glycerol, 67.5 ml Distilled Water) was used to harvest whole-cell lysates, followed by sonication. The concentration of protein in the cell lysates was estimated by using a bicinchoninic acid (BCA) assay. Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels (Invitrogen, UK) were used to separate proteins. The separated proteins were transferred by perpendicular electrophoresis to a nitrocellulose Hybond^TM C membrane (Amersham, Buckinghamshire, UK). Monoclonal Mouse Anti-Human primary antibodies Actin 1:1000 (#: A4700, Sigma-Aldrich), MDM2 1:300 (#: OP46-100UG, Merck Millipore), p21^WAF1 1:100 (#: OP64, Calbiochem), p53 1:500 (#: NCL-L-p53-DO7, Leica Microsystems Ltd.) and Polyclonal Rabbit Anti-Human primary antibody BAX 1:1000 (#: 2772S, Cell Signalling) were used. Secondary goat anti-mouse/rabbit HRP-conjugated antibodies (#: P0447/P0448, Dako) were used at 1:1000. All antibodies were diluted in 5% milk/1XTBS-Tween (w/v). Enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm) were used to visualize the proteins.

Cell lysates were analysed by SDS-PAGE using the following antibodies: PO₄-SMAD2 (Ser465/467) (rabbit polyclonal, #3101, Cell Signalling Technology [CST]), SMAD2 (mouse monoclonal, C16D3, CST), SMAD2/3 (mouse monoclonal, Clone 18, BD transduction Laboratories), SMAD4 (mouse monoclonal, B-8, Santa Cruz Biotechnology), TGFBR1 (rabbit polyclonal, V-22, Santa Cruz Biotechnology), CDKN1A (rabbit polyclonal, C19, Santa Cruz Biotechnology), RHOA (mouse monoclonal, 26C4, Santa Cruz Biotechnology), PO₄-SRC (Tyr416) (rabbit monoclonal, D49G4, CST), SRC (rabbit monoclonal, 36D10, CST), PO₄-p44/p42 MAPK (ERK1/2) (Thr202/Tyr404) (rabbit polyclonal, #9101, CST), p44/p42 MAPK (ERK1/2) (rabbit polyclonal, #9102, CST), β-actin (mouse monoclonal, AC-74, Sigma). Secondary HRP-conjugated antibodies (Dako) and enhanced chemiluminescence (GE Healthcare) was used to detect bound antibody.

The protein-based concentration of EVs, CM and Sup was measured using Pierce BCA Protein Kit (Cat # 23225, ThermoFisher Scientific) for bicinchoninic acid assay according to the protocols provided by the manufacturer. Samples (40 µg proteins) were separated on 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) by applying 120 Volt (V) in running buffer for 90 min. In the next step, proteins were transferred to the nitrocellulose membranes by applying 110 V in transfer buffer for 70 min. Membrane blocking was done with 5% skimmed milk prepared in Tris-buffered saline containing 0.1% Tween 20 (1 × TBST). Incubation of membranes with primary antibodies was performed overnight at 4 °C. Primary antibodies included: TSG101 (SC-7964, Santa Cruz Biotechnology) with 1:200 dilution, CD81 Antibody (SC-166029, Santa Cruz Biotechnology) with 1:200 dilution and CD9 (Ab92726, Abcam) with 1:1000 dilution. Afterwards, membranes were incubated for 1 h with respective secondary antibodies (HRP conjugated antibodies) (Dako, Agilent Denmark).

RPMI-1640, PBS, Glutamax, and Hepes were obtained from Lonza (Austria). Fetal calf serum was purchased from PAA (GE Healthcare, UK), MEM w/o leucine, 0.25% Trypsin/EDTA from Gibco, and YoYo-1 fluorescent dsDNA staining from Molecular Probes (Life Technologies, UK), and tritiated Leucine from Perkin Elmer (Waltham, MA). Cyclosporine A was purchased from Calbiochem (San Diego, CA) and dissolved in ethanol to 8.3 mM stock solution. The GenElute Mammalian total RNA kit and general laboratory chemicals were from Sigma Aldrich (St. Louis, MO), the Cell Titer 96 AqueousOne solution (MTS) cell proliferation assay was from Promega (Madison, WI). RT² Profiler PCR Array System, including the cDNA synthesis kit, and SYBR green were from SABiosciences (Qiagen Nordic). Chemicals for validation of gene expression were from Applied (Life Technologies, UK). Plastic ware for cell culture was from Nunc (Thermo Scientific), gels and buffers for protein electrophoresis from Life Technologies, HRP-conjugated antibodies from Dako (DK), and chemiluminescent super-signal substrate from Thermo Scientific.

Anti-human Bax rabbit polyclonal antibody (pAb) and anti-human Ku70 mouse monoclonal antibody (mAb) were purchased from BD Bioscience (Erembodegem, Belgium); another anti-human Ku70 mouse mAb for immunoprecipitation, anti-human HDAC-6 and anti-human SirT-3 rabbit (pAb) were purchased from Abcam (Cambridge, UK); anti-human Ku80 mouse mAb from Signal Transduction (USA), anti human SirT-1 and anti pan-K (Acetylated lysine) from Upstate (Upstate Biotechnology, Lake Placid, NY, USA); anti human Bcl-2 mouse mAb was purchased from DAKO (Glostrup, Denmark). Detection by immunoblotting was carried out with anti-mouse or anti-rabbit secondary HRP-conjugated Antibodies (Dako) diluted at 1: 2,000. Immunofluorescence staining was performed using anti-rabbit FITC-conjugated secondary Antibodies (Dako) diluted at 1: 50. Histone deacetylase inhibitors (HDACIs), Nicotinamide (NAM) and Trichostatin (TsA) were purchased from Wako Chemicals, Japan.

Total kidney lysates were homogenized in NP40 lysis buffer (Invitrogen, Carlsbad, USA) supplemented with protease cocktail inhibitor (Roche, Basel, Switzerland). After sonication, proteins were resolved by Tris-acetate-SDS acrylamide gel electrophoresis and transferred onto PVDF membranes (Invitrogen, Carlsbad, USA) and blocked in 5% dry milk in TBS-T (TBS pH 7.6, 0.1% Tween-20). After incubation with respective primary antibodies against PD-L1 (ab238697, Abcam, Cambridge, UK) and β-actin (ab8227, Abcam, Cambridge, UK) were used following incubation with secondary HRP-conjugated antibodies (Dako, Glostrup, Denmark). Luminescence was detected on an X-ray film using chemiluminescent substrate (Cell Signaling, Danvers, USA). Individual lanes represent biological replicates and densitometry was performed using ImageJ software (National Institute of Health, Bethesda, USA), PD-L1 band density was quantified relative to β-actin.