Anti ha rabbit pab

Anti-KSHV LANA ORF73 rat monoclonal antibody (MAb) was purchased from Advanced Biotechnologies Inc. (Columbia, MD, USA) [78 (link)]. Anti-phospho-AKT (Ser473) rabbit MAb, anti-AKT rabbit polyclonal antibody (PAb), anti-Flag M2 rabbit MAb, anti-His rabbit MAb and anti-HA rabbit PAb were obtained from Cell Signaling Technologies (Beverly, MA, USA). Anti-GRK2 mouse MAb, anti-CXCR2 rabbit PAb, anti-GAPDH mouse MAb, anti-α-Tubulin mouse MAb, and horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GFP mouse MAb was from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Western blotting analysis was performed as described previously [83 (link)]. MK-2206, an AKT inhibitor, was purchased from Selleck Chemicals (Shanghai, China).

HEK-293T cells grown in 24-well culture plates (2 × 10⁵ cells/well) were transfected with chcGAS-2HA and chSTING-3FLAG using Lipofectamine 2000. Twenty-four hours later, the transfected cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Tritonx-100 for 20 min, and blocked in 5% BSA for 30 min at RT, sequentially. The cells were next incubated with anti-HA rabbit pAb (Cell Signaling Technology, Danvers, MA, USA) and anti-FLAG mouse mAb (1:500 each) overnight at 4 °C, then the second DyLight^TM 488 Conjugated Goat Anti-rabbit IgG and DyLight^TM 594 Conjugated Goat Anti-mouse IgG (Invitrogen, Carlsbad, CA, USA, 1:800 each) at RT for 1 h. After washing with PBS, the cells were counter-stained with 0.5μg/mL DAPI for 15 min and the coverslips sealed with nail polish. Lastly, the cells were visualized under laser-scanning confocal microscope (LSCM, Leica SP8, Solms, Germany) at the excitation wavelengths 340 nm, 488 nm, and 594 nm, respectively.