Anti rabbit texas red

The following antibodies were used in immunoblots and immunofluorescence: rabbit anti-G3BP1 [14 (link)], mouse anti-G3BP1 (Bethyl Labs, Montgomery, TX, USA), rabbit anti-G3BP2 (Assay Biotech, Sunnyvale, CA, USA), goat anti-TIA1 (Santa Cruz, Dallas, TX, USA), goat anti-TIAR (Santa Cruz), anti-Rck/p54(DDX6) (gift from CE Cameron), anti-rabbit Alexa Fluor 488 (ThermoFisher, Rockford, IL, USA), anti-mouse Alexa Fluor 647 (Invitrogen), anti-rabbit Texas Red (ThermoFisher), anti-goat Texas Red (ThermoFisher).

bEnd5 cells (2.5 × 10³/well) were seeded onto glass coverslips in 24‐well plates. Cells were pre‐stimulated with VCE‐004.8 at several concentrations for 1 h and incubated for 24 h with interleukin 1 beta (IL1β) (R&D Systems, Minneapolis, MN, USA) plus tumor necrosis factor alpha (TNFα) (R&D Systems) (for VCAM1 study) or interleukin 6 (IL-6) (R&D Systems) plus TNFα (for ZO-1 study). Cells were then washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature (RT). After fixation, cells were washed twice with PBS, permeabilized with 0.5% Triton X-100 in PBS at RT for 5 min and blocked in PBS with 3% BSA for 1 h. Cells were incubated overnight at 4 °C with the following primary antibodies diluted in PBS with 3% BSA: rabbit monoclonal anti-VCAM1 (1:100, #ab134047, Abcam) or rabbit polyclonal anti-ZO-1 (1:100, #10,222,233, Invitrogen; Carlsbad, CA, USA). Next, cells were washed three times with PBS and incubated with the secondary antibody anti-rabbit Texas Red (1:100, #A-6399, Thermo Fischer Scientific) for 1 h at RT. Finally, coverslips were mounted with Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) for nuclear staining. Images were acquired using a spectral confocal laser-scanning microscope LSM710 (Zeiss, Jena, Germany) with 25 × /0.8 Plan-Apochromat oil immersion lens.