Alexa fluor 594 goat anti rabbit igg

HEK293T was obtained from ATCC, and the cell line was tested negative for mycoplasma. HEK293T cells were maintained in DMEM (#11960044, Gibco, Waltham, MA, USA) with 10% fetal bovine serum (#16140071, Gibco, Waltham, MA, USA). Plasmid transfection was conducted utilizing Effectene Transfection Reagent (#301425, Qiagen, Hilden, Germany) following the manufacturer’s guidelines.
For the luciferase reporter assay, HEK293T cells were collected 48 h post-transfection and assessed using the Dual Luciferase Reporter Assay Kit (#E1910, Promega, Madison, Wisconsin, USA), in accordance with the manufacturer’s instructions.
For microscopic analyses, HEK293T cells were seeded on micro cover glasses in wells of six-well plates 24 h before transfection. 48 h after transfection, cells were immunostained with rabbit anti-HA monoclonal (#3724S, Cell Signaling Technology, Danvers, Massachusetts, USA) at a 1:1,000 dilution, and visualized with secondary antibody, Alexa Fluor 594 goat anti-rabbit IgG (#14708S, Cell Signaling Technology, Danvers, Massachusetts, USA), at a 1:1,000 dilution. Cell nuclei were stained by DAPI (1.0 mg/ml, #564907, BD Biosciences, Dubai, UAE) for 1 min. Fluorescent images were obtained by ECLIPSE E800 microscope (Nikon, Tokyo, Japan) with a SPOT RT Slider digital camera (SPOT Imaging Solutions, Sterling Heights, MI, USA).

Cells (1 × 10⁴/well) were cultured on 96-well culture plate (Corning, USA). After incubation with different test substances, the cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min. Next, the cells were blocked with 5% BSA for 1 h and incubated with primary antibody overnight at 4 °C. Alexa-conjugated secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-rabbit IgG, Cell Signalling Technology, MA, USA) were applied and incubated at room temperature for 1 h. Cell nuclei were stained with DAPI (Cell Signalling Technology, MA, USA) for 10 min. Finally, the cells images were analysed by ImageXpress® Micro Confocal (Molecular Devices, USA).

The cells seeded and grown on glass coverslips were fixed with 4.0% PFA in PBS for 1 h at RT. After washing with PBS, the fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were incubated sequentially with 10% normal goat serum for 30 min and with primary antibodies at 4 °C overnight. The cells were washed, and stained with secondary antibodies, Alexa Fluor 594 goat anti-rat IgG or Alexa Fluor 594 goat anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA). Ultimately, nuclear staining with DAPI (Invitrogen Carlsbad, CA, USA) was carried out. The samples were visualized using a laser-scanning confocal imaging system (LSM 780 META; Carl Zeiss, AG, Jena, Germany).

Cells (1×10⁴/well) were cultured on 96-well glass culture plate (Corning, USA). After incubation with different concentrations of KYZ3, cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min. Next, the cells were blocked with 5% BSA for 1 h and incubated with the primary antibody overnight at 4 °C. Alexa-conjugated secondary antibodies (Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-rabbit IgG, Cell Signaling Technology, MA, USA) were applied and incubated at room temperature for 1 h. Cell nucleus was stained with 4, 6-diamino-2-phenyl indole (DAPI, Cell Signaling Technology, MA, USA) for 10 min. Finally, the cells images were analyzed by ImageXpress® Micro Confocal (Molecular Devices, USA).