Irdye goat anti mouse

The Western blot procedure was consistent with our previous work and detailed elsewhere (Hüttemann et al., 2012 (link), 2013 (link); Lee et al., 2012 (link); Malek et al., 2013 (link)). Samples were loaded onto 7.5% (TSP-1, PGC-1α, PGC-1β, and ADAMTS-1) or 12% TGX pre-cast gels (Bio-Rad, Hercules, CA, USA).
The mouse monoclonal primary antibodies used were TSP-1 (1:500, sc-59886, Santa Cruz Biotechnology, Inc), CD47 (1:500; 3847-1, Epitomics), PGC-1β (1:100; sc-373771, Santa Cruz Biotechnology, Inc), α-tubulin (1:2,000, ab11304, Abcam), ADAMTS1 (1:500; sc-47726, Santa Cruz Biotechnology, Inc), and GAPDH (1:2,000, ab9484, Abcam). The polyclonal primary antibodies used were VEGF (1:500, sc-507, Santa Cruz Biotechnology, Inc), VEGFR2 (1:500; 2479, Cell Signaling), FoxO1 (1:200; 2880, Santa Cruz Biotechnology, Inc), Anti-TFAM (1:1,000; ab131607, Abcam), and PGC-1α (1:1,000; AB3242, Millipore). The secondary antibodies used were goat anti-mouse IRDye (1:30,000) and goat anti-rabbit IRDye (1:30,000) purchased from Li-Cor Biosciences. Loading control for target proteins were normalized to α-tubulin or GAPDH. Quantification of bands were analyzed with the Odyssey software program (Li-Cor Biosciences).

Approximately 5 µg of GST or GST-tagged protein fragments were incubated in Glutathione-Sepharose beads (GE Healthcare) in GST-lysis buffer at 4°C overnight. The bead-bound proteins were then incubated in GST-lysis buffer containing 5% BSA at 4°C for 2 hr. Meanwhile His-tagged full-length protein (~50 µg) was pre-cleared in Glutathione-Sepharose beads. The pre-cleared protein was then incubated with bead-bound proteins in Phosphate Buffered Saline containing 0.1% Tween 20 (PBST). After 2 hr incubation, the beads were washed three times with PBST containing 500 mM KCl and eluted with 2x-SDS loading buffer. The pull-downs were then analyzed by SDS-PAGE and subsequent Coomassie staining. For specific detection of His-tagged proteins, anti-His (1:1000) (Abcam) was used. Bound primary antibodies were detected using Goat anti-Mouse IRDye (1:10000) using an odyssey imaging system (Li-Cor).

Puromycin-selected HeLa or COS-1 cells were seeded in 6-well plates and transfected with 0.3 ug NL4-3 (HeLa) or 1 ug pSARMX proviral vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. On day 2 post-transfection, cell pellets and culture supernatants were harvested. Culture supernatants were clarified by low speed centrifugation and filtered through a 0.2 µm filter, then layered onto a 20% sucrose cushion in PBS and centrifuged at 20,000 g for 2 hours at 4°C using Sorvall Discovery M-150 SE micro-ultracentrifuge (Thermo Electron Corporation, Waltham, MA, USA). Virion pellets and corresponding cell pellets were dissolved in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Virion and cell lysates were separated on 12% polyacrylamide gels and subjected to Western blotting. HIV-1 Gag detection was performed with mouse anti-p24 monoclonal CA-183 antibody (provide by Bruce Chesebro and Kathy Wehrly through the NIH AIDS Research and Reference Reagent Program). Rabbit anti-p27 polyclonal antibody [41] (link)was employed for detection of M-PMV Gag. IRDye goat anti-mouse and IRDye goat anti-rabbit secondary antibodies used for Western blots were obtained from Li-cor Biosciences (Lincoln, NE, USA). Blots were developed and analyzed using the Li-Cor Odyssey infrared detection system.

Intracellular MMP‐9 protein contents in PF were determined by immunoblot. Day 7 PF plated in T‐25 tissue culture flasks were treated with 0.1–100 ng/mL MCP‐1 (Thermo Scientific, Rockford, IL) overnight or were treated with buffer alone. Following treatment, cells were scraped in 4°C PBS, and obtained protein lysates were resuspended in Laemmli sample buffer, sonicated, heat‐denatured, and electrophoresed in 7–15% tris HCl Ready Made gels (Biorad, Hercules, CA). Resolved proteins were transferred to a PVDF membrane (Immobilon/Millipore, Bedford, MA). Membranes were blocked using Odyssey Blocking Buffer (Li‐Cor Biotechnology) and incubated with a rabbit monoclonal antibody against MMP‐9 (1:1000) (Abcam, Cambridge, MA) followed by a 680 IRDYE goat anti‐rabbit secondary antibody (1:8000) (Li‐Cor Biotechnology) and imaged using the Odyssey Imaging System fluorescence scanner (Li‐Cor Biotechnology). After restoring the membranes, they were incubated with a commercial mouse monoclonal antibody against beta actin (1:5000) followed by 800 IRDYE goat anti‐mouse (1:8000) (Li‐Cor Biotechnology). In a separate set of experiments, Day 7 PF were treated with BAPTA/AM (10 μmol/L), MCP‐1 (10 ng/mL), BAPTA/AM + MCP‐1, or ionomycin (10 μmol/L) (Calbiochem, San Diego, CA) for 10 min before harvesting cells and following the protocol above.

The parasite proteins were extracted in reducing sample buffer for SDS-PAGE. Five micrograms of recombinant PvRALP1-Ecto or PvRALP1-Tr protein were loaded into each well and separated by SDS-PAGE under reducing conditions. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) in a semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). After blocking with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 (PBS-T), the membranes were probed with mouse anti-PvRALP1-Ecto and anti-PvRALP1-Tr sera, rabbit anti-PvRALP1-Tr serum, anti-GST monoclonal antibody (Novagen, Madison, WI, USA), anti-penta-His monoclonal antibody (Qiagen), preimmune mouse serum, pooled sera from P. vivax malaria patients or noninfected individuals, all diluted 1:200 in PBS-T. IRDye goat anti-mouse, IRDye goat anti-rabbit, or IRDye goat anti-human sera (LI-COR Biosciences, Lincoln, NE, USA) were used to detect recombinant proteins according to the manufacturer’s instructions. Data were scanned with an Odyssey infrared imaging system (LI-COR Biosciences) and analysed with Odyssey software (LI-COR Biosciences).

Recombinant proteins were analyzed by 13% SDS-PAGE under reducing conditions. The separated proteins were transferred onto a 0.45-μm PVDF membrane (Millipore) in semidry transfer buffer (50 mM Tris, 190 mM glycine, 3.5 mM SDS, 20% methanol) at a constant 400 mA for 40 min using a semidry blotting system (ATTO Corp., Tokyo, Japan). The PVDF membrane (Cytiva, Marlborough, MA) containing recombinant protein was blocked with 5% skim milk in PBS-T (0.5% v/v Tween-20 in 1× PBS) and then incubated with anti-GST antibody (Novagen, Reno, NV) and mouse and rabbit immune sera diluted 1:2,000 in PBS-T. After the primary antibody reaction, the membrane was incubated with the secondary IRDye^® goat anti-mouse (1: 5,000 dilution) or IRDye^® goat anti-rabbit (1: 5,000) (Li-COR^® Bioscience, Lincoln, NE) antibodies to detect antigens. The results were visualized in the Odyssey infrared imaging system and analyzed with Odyssey software (Li-COR^® Bioscience) (Lee et al., 2016 (link)).