Anti gas6 antibody

Receptor tyrosine kinases (RTKs) were inhibited in U2OS cells overnight with 1 μM of each of the following inhibitors: Gefitinib (#HY-50895), Lapatinib (#HY-50898), Bemcentinib (#HY-15150), BMS-536924 (#HY-10262) purchased from MedChemExpress; Erdafitinib (#HY-18708) Lenvatinib (#HY-10981), Cabozantinib (#HY-13016), Galunisertib (#HY-13226), Linsitinib (#HY-10191) from Cedarlane Laboratories; and Crizotinib (#PF-02341066) from Selleckchem.com. PI3K was inhibited by treating cells with LY294002 (Millipore Sigma; #440202) at 10 μM for at least 1 h. Gas6-mediated stimulation of AXL in U2OS cells was performed by serum-starving cells for 24 hours prior to the addition of 200 ng/mL recombinant Human Gas6 (R&D Systems; #885-GSB) to the culture medium for 30 min. For AXL receptor blockade, U2OS cells were incubated with 20 μg/mL anti-AXL antibody (R&D Systems; #AF154) or 50 μg/mL anti-GAS6 antibody (R&D Systems; #AB885). Normal goat polyclonal IgG (R&D Systems; #AB-108-C) was used as control (50 μg/mL). Rho-associated kinases (ROCK) were inhibited with Y-27632 (Cell Signaling Technology; #13624) at 1 or 5 μM.

Cells cultured in 96-, 24-, or 6-well plates were infected with ZIKV at different MOIs for 1 h at 37°C. The HTO were infected at 10⁵ PFU as described previously (38 (link)). For Axl receptor blockade, SC were preincubated with 10 μg/ml anti-Axl antibody (catalog no. AF154; R&D Systems) or 50 μg/ml anti-Gas6 antibody (catalog no. AB885; R&D Systems), or goat polyclonal IgG control (catalog no. AB-108-C; R&D Systems) for 2 h at 37°C prior to infection. Following infection, wells were replenished with fresh media without antibody. For Gas6 depletion, SC were serum deprived for 3 h prior to infection, infected without serum or in the presence of 5 nM recombinant human Gas6 (rhGas6) (catalog no. 885-GSB; R&D Systems), and then replenished with media containing 5% fetal bovine serum (FBS) following 1-h infection. For Axl kinase inhibition experiments, SC or HTO were preincubated with 1 μM R428 (catalog no. BGB324; Selleckchem) for 2 h prior to infection and after infection replenished with fresh media containing 1 μM R428 or DMSO vehicle control (1:10,000 dilution). ZIKV titers in the culture supernatant and intracellular ZIKV RNA was quantified by plaque assay and quantitative reverse transcription-PCR (qRT-PCR), respectively, as reported previously (48 (link), 49 (link)).