Polyclonal goat anti rabbit immunoglobulins hrp

Whole-cell extract was used for Western blotting by lysing cells in RIPA buffer (Sigma-Aldrich, Life Science) supplemented with protease inhibitors. Protein was quantified using the PierceTM BCA Protein Assay Kit (ThermoScientific). Primary antibodies for HIF2A (Novus Biologicals NB100-122), FLAG (Sigma #F3165), VHL (BD Biosciences #564183) and β-actin (Sigma #A1978) were used. Secondary antibodies were polyclonal goat anti-mouse immunoglobulins/HRP (Dako) and polyclonal goat anti-rabbit immunoglobulins/HRP (Dako).

Transfected Bm5 and High Five cells were collected and lysed by sonication (Branson) in a lysis buffer consisting of Tris-Cl 50 mM, NaCl 150 mM, EDTA 1 mM, Triton-X-100 1%, Sodium Dodecyl Sulphate (SDS) 0.5% and Protease Inhibitor Cocktail Tablets Complete (Roche). Next, the total amount of proteins was quantified by means of a Bicinchoninic acid assay, after which 18 μg of each sample were separated by SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a Trans-Blot Turbo Mini PVDF membrane using the Trans-Blot Turb Blotting System (Bio-Rad). The blots were washed and blocked with a skimmed powder milk solution 5% for 2 hours. Anti-c-Myc antibodies produced in rabbit (Sigma-Aldrich) were diluted (1:5000) and incubated with the blots overnight at room temperature. Washing was then performed, followed by 2 hours of incubation with Polyclonal Goat Anti-Rabbit Immunoglobulins/HRP (Dako), diluted 1:50000. Finally, the blots were washed and the detection was performed with Super Signal West Dura Extended Duration Subtract Kit (Thermo Scientific). The chemiluminescent bands were visualized using a ChemiDoc™ MP Imaging System with Image Lab Software (Bio-Rad).

Interleukin-1β (IL-1β) was obtained from Peprotech (Rocky Hill, NJ). Polyclonal rabbit antibody against COX-2 was from Millipore (Temecula, CA). Polyclonal goat anti-rabbit Immunoglobulins/HRP were from Dako (Glostrup, Denmark). Diaminobenzidine (DAB) was from Vector (Burlingame, CA). Monoclonal antibodies against phospo-NFκB p65 (Ser536), phospho-p38 (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), and RelB were from Cell Signaling Technology (Beverly, MA). 12-O-tetradecanoyl phorbol 13-acetate (TPA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other reagents were from Sigma-Aldrich (St. Louis, MO).

Isolated neutrophils were resuspended in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitor mixture (both Cell Signaling) followed by cell sonication. Samples were separated on SDS-PAGE and transferred to nitrocellulose membranes (Peqlab, 0.2 μm). The following antibodies were used: mouse monoclonal anti-human Mcl-1 (BD Pharmingen, 559027), rabbit polyclonal anti-human Mcl-1 (phospho S159; Abcam, ab111574), rabbit polyclonal Phospho-Erk1/2 (Thr202/Tyr204, Cell Signaling, 9101), rabbit polyclonal Erk 1/2 (Cell Signaling, 9102), rabbit monoclonal Akt (Cell Signaling, 4691), rabbit polyclonal Phospho-Akt (Ser473) (Cell Signaling, 9271), monoclonal mouse anti-human Serpin A1/α1-Antitrypsin (R&D Systems, MAB1268), mouse monoclonal GAPDH (Novus Biologicals, NBP2-27103). The membranes were further incubated with polyclonal goat anti-mouse immunoglobulins/HRP (Dako, P0447) or polyclonal goat anti-rabbit immunoglobulins/HRP (Dako, P0448), respectively. For detection of human immunoglobulin, a HRP-conjugated goat-anti human IgG + IgA + IgM (H+L) antibody (Biozol, MBS539207-2) has been used. Bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32209) and UptiLight HRP Blot Chemiluminescent ECL Substrate (Uptima, UP99619A).

Antibodies used for flow cytometry studies included an SSPE serum (from a 1978 Belfast case), anti-MV-H mAbs 55 (SLAM-binding site) [19 (link)], BH129 (anti-NE epitope) [20 (link)], BH216 (anti-Noose epitope) [21 (link)], and anti-MV-F mAb Y503 (a gift from D. Gerlier, Inserm U758). Secondary antibodies were rabbit anti-human IgG and goat anti-mouse IgG (both from Millipore). For the western blot an anti-MV-H mAb BH195 [20 (link)] and an anti-MV-F mAb (a gift from Christian Buchholz, Paul Ehrlich Institute, Langen, Germany) were used together with secondary antibodies, polyclonal rabbit anti-mouse immunoglobulins/HRP, and polyclonal goat anti-rabbit immunoglobulins/HRP, respectively (Dako). Antibodies used for the confocal microscope studies were anti-MV-H mAb BH129, anti-MV-F mAb Y503, and anti-MV-M mAb 8910 (Millipore).

Western blot analyses of ITGA4 and beta tubulin proteins were performed on approximately 20 µg of protein using anti-ITGA4 antibody (Cell Signaling Technology, cat. no. 4600)^{36 (link)} at 1:1000 dilution and rabbit polyclonal anti-β-tubulin (Invitrogen™, cat. no. PA1-41331)^{37 (link)} at 1:1000 dilution overnight at 4 °C. Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako, cat. no P0448)^{38 (link)} at a dilution of 1:10,000 and Luminata Crescendo western HRP substrate were used for immunodetection. The blots were exposed for a serial scan of 30 s using the Fusion FX system (Vilber Lourmat, Marne-la-Vallée, France) and the entire image was processed. Quantification was performed using Image J (Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).

Protein gel electrophoresis was performed by loading 5 μg of cell lysate per lane on a 4-12% Bis-Tris NuPAGE gel (Invitrogen) followed by transfer to a PVDF membrane (Invitrogen). The membrane was blocked with 5% Milk/TBS (0.1%Tween20), prior to overnight incubation with primary antibody incubation at 4°C. Secondary antibody probing (Polyclonal Goat Anti-Rabbit Immunoglobulins/HRP; Dako; Carpinteria, CA) was done for 1 hour at room temperature. Results were visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare; Pittsburg, PA) and Amersham Hyperfilm ECL (GE Healthcare). Reprobing to verify loading control was done after rinsing the membrane and reblocking before subsequent antibody probing. BCL-XL (clone 54H6), GAPD (clone 14C10), and beta-actin (clone 13E5) antibodies for immunoblotting were obtained from Cell Signaling Technologies (Danvers, MA).