Hrp conjugated streptavidin

The sγc in supernatants of cultured cells were immunoprecipitated with α-mouse IL-2Rγ antibody (R&D systems) and protein A/G agarose beads (Santa Cruz Biotechnology). Immunoprecipitates were resolved by SDS-PAGE (Novex) under reducing by dithiothreitol (DTT) or non-reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Blots were incubated with biotinylated α-mouse IL-2Rγ antibody (R&D systems), followed by HRP-conjugated streptavidin (BioLegend). The membranes were developed by enhanced chemiluminecence (ECL) reagents (GE Healthcare). The bands were detected using LAS-3000 Imaging system (Fujifilm).

Gas6, IL-10 and TNF-α levels were measured in supernatants of cell cultures using sandwich ELISA according to standard procedure [32 (link)]. Briefly, 96-well plates were precoated overnight with a capture antibody. Samples from cell culture supernatants were applied to precoated plates in duplicate. Serial dilutions of purified recombinant rhGas6 (R&D Systems) were used to construct a standard curve. Blank wells received serum-free X-Vivo™15 medium. A purified goat polyclonal anti-human Gas6 antibody (R&D Systems) was used for capture. Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection. The plates were developed with 3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped with 2 N sulfuric acid. Absorbance was detected at 450 nm and read with a reference wavelength set at 570 nm using a VersaMAX ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density for each point was the average of duplicate samples. Concentrations were determined using SoftMax software (Molecular Devices) by applying four-parameter logistic regression to the standard curve. IL-10 and TNF-α levels were measured using human IL-10 ELISA MAX Standard kit and TNF-α ELISA MAX Standard kit (Biolegend), following the manufacturer’s instructions.

Splenocytes were suspended in 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin–streptomycin (Thermo Fisher) at a final concentration of 1 × 10⁷ cells/mL. Then, 1 × 10⁶ cells were added to each well of a 96-well plate (Corning Inc., Corning, NY, USA). With 10 μL of PMA + ionomycin (stock solution concentrations: 500 ng/mL + 10 μg/mL; DAKEWE, Beijing, China) as positive control, gE was added to each well at a final concentration of 10 μg/mL. After incubation at 37 °C for 24 h in a 5% CO₂ atmosphere, the supernatants of the cells were collected to test the contents of IL-2 and IFN-γ. Briefly, anti-IL-2 (3 μg/mL) and anti-IFN-γ (4 μg/mL) capture antibodies (Invitrogen, Carlsbad, CA, USA) dissolved in PBS were added to coat 96-well plates and incubated overnight at 4 °C. After blocking with 5% (w/v) skim milk at 37 °C for 1 h, 50 μL of cell supernatant was added to each well and incubated for another 3 h at room temperature. PBS-dissolved standard mouse IL-2 and IFN-γ proteins (PeproTech, Cranbury, NJ, USA) were used to generate standard curves. Biotin-conjugated detection antibodies specific for IL-2 or IFN-γ (2 μg/mL, Invitrogen, USA) and HRP-conjugated streptavidin (1 μg/mL, BioLegend, San Diego, CA, USA) were then added and incubated for 1.5 h. The results were detected as mentioned above in the ELISA analysis.