Irdye 680cw goat anti mouse antibody

T-47D cells grown at 1×10³ cells per well in 96-well plates (Corning Incorporated Costar, #3603). After 24 h incubation, chemical compounds (10 μM) were added to each well. After 48 h incubation, plates were fixed with 4% paraformaldehyde (PFA) for 20min at room temperature (RT). Then the cells were permeabilized with 0.2% Triton X-100 for 15min at RT, and blocked with Odyssey Blocking Buffer (TBS) (LI-COR, 9275000) for 1 h at RT. The cells were incubated at 4°C overnight with a rabbit monoclonal anti-ORF1p antibody (diluted 1:200, Abcam) and a mouse monoclonal anti-beta-actin antibody (diluted 1:250, Abcam) with gentle shakes. After five washes with PBS, the cells were stained with an IRDye^® 800RD Goat anti-Rabbit antibody (diluted 1:400, LI-COR, #926–68071) and IRDye^® 680CW Goat anti-Mouse antibody (1:600, LI-COR, #926–32210) at RT for 2 h. The microplates, were scanned with the Odyssey system (LI-COR), and the integrated fluorescence intensities representing the protein expression levels were acquired using Image Studio Ver 5.2 software (Egorina et al., 2006 (link)). Compared with DMSO control, compounds with an inhibition rate over 50% were defined as promising compounds.

Protein concentration in cell extracts was measured by the BCA Protein Assay Kit (Cole‐Parmer). Forty micrograms protein/lane was electrophoretically separated on a 10% SDS polyacrylamide gel and electrically transferred onto a Hybond‐P PVDF transfer membrane (Millipore) for 1.5 h. Membranes were blocked in PBS‐milk 5% at RT for 1 h. Next, membranes were incubated in block solution overnight with the following primary antibodies: rabbit anti‐lamin‐B1 (1:1000; Abcam) and mouse anti‐GAPDH (1:1000; Abcam). Membranes were incubated for 1 h with IRDye 680CW goat anti‐mouse antibody and IRDye 800CW goat anti‐rabbit antibody (LI‐COR, 1:20,000) and then scanned and analyzed using Un‐Scan‐It gel version 6.1 (Silk Scientific).