Mouse anti myc antibody

Immunoprecipitation of expressed proteins was performed using lysates from HEK293 cells transfected with indicated constructs. One day after transfection, cells were lysed in lysis buffer (50mM HEPES, pH 7.4 containing, 150mM NaCl, 1.5mM MgCl2, 0.5mM CaCl2, 10% (v/v) glycerol, 1% (v/v) Triton-X100, 1mM PMSF, and 0.5mg/ml each of leupeptin, bestatin, pepstatin) with rocking for 30 minutes. Lysates were cleared using Protein-G sepharose beads (GE Healthcare) and incubated overnight with 6-8μg rabbit anti-GFP antibody (Novus Biologicals, NB 600-303) or mouse anti-myc antibody (Sigma M4439). Immunoprecipitation of endogenous protein was performed using MCF-7 cells. Cells were grown to confluence and lysed for 30 minutes. Lysates were cleared using Protein-G sepharose and incubated overnight with 10μg mouse anti-ERR1 antibody (Abcam ab418618). Other primary antibodies include mouse anti-GFP (Roche, 1814460), rabbit anti-myc (Cell Signaling, 2276S), goat anti-ERRα (Santa Cruz Biotechnology, sc-32971) and goat anti-KIF17 IgG (M20, Santa Cruz).

To determine the cellular localization of the S protein of PEDV and the TTSPs, Vero cells were transfected with pCMV-Myc plasmids expressing TMPRSS2, HAT, DESC1, or MSPL, or with empty plasmid serving as a negative control. At 24 h post-transfection, the cells were washed with PBS and infected with PEDV LJB/03 at an MOI of 1. The pCMV-Myc-transfected cells were infected with PEDV in the absence or presence of 3 μg/mL trypsin. At 24 h post-infection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 0.3% bovine serum albumin. Then, the cells were incubated with mouse anti-Myc antibody (Sigma) and rabbit anti-PEDV S protein polyclonal antibody (developed in our laboratory) at RT for 1 h. After washing with PBS three times, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ZSGB-BIO) and Alexa Fluor 647-labeled goat anti-mouse IgG (H + L) (ZSGB-BIO) secondary antibodies at RT for 1 h. After washing, the cells were treated with DAPI (Beyotime). The coverslips were mounted on glass microscope slides in mounting buffer and examined using a laser scanning microscope (Leica TCS SP2, Wetzlar, Germany). Further image analysis, including calculation of the Pearson correlation coefficient (PCC), was performed with Image J with Just Another Colocalization Plugin [32 (link),42 (link)].

ELISA was performed using V_HHs secreted in the media. 600 µL of 2xTY with 100 µg/mL ampicillin and 1% glucose was added to each well of 2-mL-deep 96-well plates, each of which was inoculated with 6 µL of the master stock. This was incubated for 2 hr at 37°C with shaking, after which 1 mM isopropyl thiogalactoside (IPTG) was added. The plates were then shifted to 30°C and incubated overnight. The cultures were centrifuged at 2800 rpm for 10 min at RT, and the supernatant, which contains secreted V_HHs, was carefully transferred to a fresh 96-well plate. After this, standard ELISA, as outlined in the above protocol (ELISA using phages) was carried out, using mouse anti‐Myc antibody (Sigma‐Aldrich) at a dilution of 1:1500 and goat anti‐mouse antibody conjugated to HRP (Life Technologies, USA) at a dilution of 1:1000.