Horseradish peroxidase hrp conjugated anti mouse secondary antibody

Total protein was extracted by lysis buffer and isolated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked with primary anti active β-catenin antibody (Millipore, Bedford, MA, USA) overnight at 4 °C and incubated with anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody (Cell Signaling Technology, Boston, MA, USA) after washed with Tris-buffered saline containing 10 mM Tris–HCl, 50 mM NaCl and 0.25% Tween 20 at 37 °C for 1 h. Protein quantification was performed using an enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA). GAPDH was used as a loading control.

Secretion assays were conducted as described previously (16 (link)). In brief, strains were grown for 3 h with arabinose added to 0.4% to induce fusion gene expression. Cultures were centrifuged and pellets stored for sonication as cell lysate samples. Meanwhile, supernatants were passed through 0.22-μm filters. Trichloroacetic acid (TCA) solution was added to a final concentration of 20% and placed at –20°C overnight. Supernatants were then centrifuged at 15,000 × g for 20 min at 4°C, pellets were washed with 100% acetone, and the resultant pellet was resuspended in SDS-loading dye for analysis by Western blotting.
As described previously (15 (link), 53 (link)), proteins were resolved by polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, blocked with 5% skim milk in TBST buffer (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6) for 1 h at room temperature, and then incubated with primary antibodies overnight at 4°C. Blots were washed 3 times in TBST, incubated with anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology) for 1 h, and then detected using ECL solution (Bio-Rad). Monoclonal antibodies to the V5 epitope tag and RpoB, the beta subunit of RNA polymerase used as a cytoplasmic control, were purchased from GeneTex and NeoClone, respectively.

BiTE/TriTEs were detected by immunoblotting with anti-C-terminal-His antibody (3D5, Invitrogen, UK, #R930–25). For western blotting analysis, supernatants were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. For dot blot analysis (Additional file 1), two-fold serial dilutions of the supernatants were applied directly to a nitrocellulose membrane. To generate a standard curve, a deca-His-tagged standard protein of known concentration was serially-diluted and applied in parallel. Membranes were probed with an anti-C-terminal-His tag (1:5000, clone 3D5, Invitrogen, UK, #46–069) primary antibody, then a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:3000, Cell Signalling Technology, UK, #7076). SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, UK, #34075) was applied and the membrane exposed to X-ray film, which was developed in an automatic film processor (Agfa CP1000).

Doxycycline, hydroxypropyl-β-cyclodextrin, poloxamer 407, poloxamer 188, VEGF-C and lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO, USA). Antibodies included, anti-LYVE-1, anti-VEGF receptor 3 (VEGFR3) (abcam, Hong Kong, China), anti-Akt, anti-phosphorylated Akt, anti-nuclear factor-kappaB (NF-κB) p65, anti-phosphorylated NF-κBp65, anti-IκB-α, anti-eNOS, anti-phosphorylated eNOS, anti-β-actin, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRP-conjugated anti-rabbit secondary antibody, Alexa Fluor 488-coupled goat anti-rat secondary antibody and Alexa Fluor 555-coupled goat anti-rabbit secondary antibody (Cell Signaling Technology, Inc., Danvers, MA).

The antibodies, including anti-lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) polyclonal, anti-VEGF-C monoclonal, anti-VEGF receptor 3 (VEGFR3) monoclonal, anti-CD11b monoclonal, anti-F4/80 monoclonal, and Anti- proliferating cell nuclear antigen (PCNA) monoclonal, were from abcam (Hong Kong, China). And the other antibodies, including anti-nuclear factor-kappa B (NF-κB) p65 monoclonal, anti-phosphorylated NF-κB p65 monoclonal, anti-β-actin monoclonal, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRP-conjugated anti-rabbit secondary antibody, Alexa Fluor 488-coupled goat anti-rat secondary antibody, Alexa Fluor 555-coupled goat anti-rabbit secondary antibody, were from Cell Signaling Technology Inc. (Danvers, MA). Recombinant HMGB1 (rHMGB1) and Box-A (a specific antagonist of HMGB1) were purchased from IBL International (Hamburg, Germany). VEGF-C was from Sigma (St. Louis, MO, USA).

The antibodies, including polyclonal anti-lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), monoclonal anti-Akt, monoclonal anti-phosphorylated Akt, monoclonal anti-eNOS, and monoclonal anti-phosphorylated eNOS antibodies, were obtained from Abcam (Hong Kong, China). Other antibodies, including the monoclonal anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRP-conjugated anti-rabbit secondary antibody, and Alexa Fluor 555-conjugtaed goat anti-rabbit secondary antibody, were purchased from Cell Signaling Technology Inc. (Danvers, MA). Recombinant mouse IL-33 was purchased from Alexis Biochemicals (San Diego, USA). VEGF-C was obtained from Sigma (St. Louis, MO, USA). Wortmannin and N^G-Monomethyl-L-arginine (NMA) were obtained from Abcam (Hong Kong, China).

PC3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured with RPMI 1640 culture medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% v/v fetal bovine serum (FBS) (Internegocios, Buenos Aires, Argentina), penicillin 100 U/mL, streptomycin 100 µg/mL, and amphotericin 0.5 µg/mL. Hemin was obtained from SIGMA-Aldrich (St. Louis, MO, USA). For treatments, cells were incubated 24 h in RPMI media containing 10% FBS and antibiotics and then were exposed to Hemin (80 µM, 24 h). Thapsigargin was obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS). For treatments, cells were incubated in complete RPMI media and treated with Thapsigargin 0.1 µM or 0.25 µM for 24 h. Interferon gamma (INFγ) protein was obtained from ImmunoTools (Friesoythe, Germany). PC3 cells were treated with INFγ, 500 U/mL, for 18 or 24 h.
Mouse anti-human HO-1 monoclonal antibody and rabbit anti-MX1 antibody were obtained from Abcam (Cambridge, UK). Mouse anti-human β-actin antibody, rabbit anti-LC3, and rabbit anti-GADPH antibodies were obtained from Cell Signaling (Danvers, MA, USA). Horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody was obtained from Cell Signaling. Secondary antibodies conjugated to Alexa 555 and Alexa 647 fluorophores were obtained from Molecular Probes, Invitrogen.