Td 20 20n luminometer

An hemagglutinin (HA) tag was engineered and added to the extracellular loop of CLC5 between transmembrane domains B and C (Dutzler et al. 2002) to track its intracellular trafficking. Previous studies have shown that the HA epitope tag at this location does not interfere with ClC‐5 function (Schwake et al. 2001).
Surface protein labeling of oocytes expressing CLC‐5 and CLC‐5 mutants was performed according to the previously established method described by our group (Chang et al. 2008). At 4°C, oocytes were fixed with 4% paraformaldehyde in ND96 for 15 min, washed, and incubated in 1% bovine serum albumin (BSA)‐ND96 blocking solution for 30 min. Oocytes were labeled with a primary antibody (1:200 dilution, monoclonal rat‐α‐HA 1° antibody [Roche]) for 60 min and then with a secondary antibody (1:2000 dilution, horseradish peroxidase conjugated goat‐α‐rat IgG [Jackson Labs]) for 30 min in 1% BSA‐ND96 blocking solution. Labeled oocytes were washed several times and incubated in ND96 for 10 min before exposure to 50 μL of the premixed SuperSignal ELISA Femto substrate solution (Pierce Scientific) at room temperature. Chemiluminescence was measured from single oocytes in a microcentrifuge tube using a TD‐20/20n luminometer (Turner BioSystems).

The activation of NF-κB was measured by the luciferase reporter assay. In short, cells were co-transfected with 1000 ng of the NF-κB reporter plasmid and 80 ng of the control plasmid (RL-TK, to quantify transfection efficiency). Both plasmids were kindly provided by Professor Chunguang Yan, Southeast University, China. Different concentrations of LPS were added to the cell cultures 48 h after transfection, and luciferase activity was measured using a dual luciferase reporter kit (Promega, E1910, Madison, WI, USA) by a TD 20/20n luminometer (Turner Biosystems, San Jose, CA, USA) 24 h later.