Anti rabbit antibodies

n-Hexane, chloroform, ethyl acetate (EtOAc), n-butanol, ethanol, and methanol were obtained from OCI (SEL, KR). Folin–Ciocalteu reagent (for total phenolics), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), dimethyl sulfoxide-d6 (DMSO, exclusive solvent for NMR), mushroom tyrosinase (EC 1.14.18.1), and ascorbic acid were obtained from Sigma Aldrich (St. Louis, MO, USA). All reagents used were of analytical grade. Phospho-CREB (p-CREB), phospho-PKA (p-PKA), AMPK, p-AKT, AKT, p-mTOR, mTOR, Becline, and LC3B were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against tyrosinase (TYR), TRP-1, TRP-2, microphthalmia-associated transcription factor (MITF), CREB, PKA, α-melanocyte-stimulating hormone (α-MSH), and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish-peroxidase-conjugated anti-mouse, anti-goat, and anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu reagent (for total phenolics), dimethyl sulfoxide-d₆ (DMSO), mushroom tyrosinase (EC 1.14.18.1), and ascorbic acid were obtained from Sigma Aldrich (St. Louis, MO, USA). All reagents used were of analytical grade. Phospho-CREB (p-CREB) and phospho-PKA (p-PKA) were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against tyrosinase (TYR), TRP-1, TRP-2, microphthalmia-associated transcription factor (MITF), CREB, PKA, α-melanocyte-stimulating hormone (α-MSH), and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase-conjugated anti-mouse, anti-goat, and anti-rabbit antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

Immunofluorescence assay (IFA) was done as described previously7 (link). Briefly, RK-13 cells infected with T. hominis were grown on coverslips until confluent and then fixed in acetone/methanol 50:50 v/v at −20 °C for 2 h. After blocking with 5% milk in PBS, slides were incubated for 1 h with a 1% (w/v) milk/PBS solution containing the relevant antiserum, washed in PBS and then incubated for 1 h with the appropriate secondary antibodies: Rat anti-ThHsp70 was used as a mitosomal marker, the remaining antibodies against T. hominis proteins were raised in rabbit. Goat anti-rat or anti-rabbit antibodies (Invitrogen) conjugated to Alexa 594 (red) or 488 (green) fluorophores were used as secondary antibodies. Cells were incubated with DAPI for 5 min to visualize host cell and parasite DNA. All cells were visualized using either a Leica TCS SP2 UV confocal microscope or a Zeiss Axioimager II epifluorescence microscope with a X63 objective lens.

For intracellular staining, cells were fixed and permeabilized with the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained with antibodies against IRF4 (200×; 3E4; eBioscience) or activated caspase-3 (C92-605; BD) for 30 min on ice. For assessment of the dead sub-G0 population, cells were incubated with 50 μg/mL propidium iodide (PI) in the presence of 100 μg/mL RNaseA. For pERM staining, cells were fixed and permeabilized with Cytofix/Cytoperm buffer (BD) and washed twice with Perm/Wash buffer (BD). The cells were stained with an antibody against pERM (250×; 48G2; Cell Signaling) for 40 min at room temperature. Primary antibody was detected with anti-rabbit antibodies (Invitrogen). All flow cytometry data was acquired on a FACS Canto (BD) and analyzed with FlowJo (TreeStar) software.

CRC cells were lysed with RIPA buffer (100 mM TRIS-HCl, pH 7.5, 300 mM NaCl, 0.2 % SDS with 1 % IGEPAL CA-630 and the Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific)) with subsequent centrifugation (18,000 x g, 4 °C, 20 min). Total protein concentration in lysate was quantified by BCA method (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples were loaded and separated by SDS-PAGE (Mini-PROTEAN TGX Stain-Free Gels; Bio-Rad Laboratories, CA, USA) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). NMU protein was detected with rabbit anti-NMU antibody (Genetex, CA, USA), for ERK1/2 kinases analysis mouse anti-ERK1/2 (Santa Cruz Biotechnology, USA) and rabbit anti-pERK1/2 antibodies (Invitrogen) were used. In EVs (extracellular vesicles) purity analysis we used rabbit antibodies anti-GM130, anti-annexin V and anti-flotilin-1 (Cell Signaling Technologies, Danvers, MA, USA). Proteins used as loading controls were detected by mouse anti-β-actin, mouse anti-α-tubulin or rabbit anti-GAPDH antibody (Abcam, Cambridge, GB). We used secondary HRP-conjugated goat anti-mouse (Santa Cruz Biotechnology) or anti-rabbit antibodies (Invitrogen). The signal was detected by chemiluminescence (Thermo Fisher Scientific) with Kodak BioMax Light Film from Eastman Kodak (NY, USA).