Horseradish peroxidase hrp labeled anti rabbit

BJ cells were cultured in the 6-well plate at a density of 5 × 10⁵ cells/mL. After treatment with P.g. LPS (1 μg/mL) for 12, 24, and 48 h, the cells were collected for experiments. A set of the BJ cells was pretreated with CA-074Me (50 μM) for 1 h, and then P.g. LPS (1 μg/mL) was added to the medium. The cells continued to be cultured for 48 h and were then harvested.
The cells were electrophoresed in 7.5% or 12% SDS-polyacrylamide gels, and the proteins on the SDS gels were transferred electrophoretically to nitrocellulose membranes, which were washed with PBS and then blocked for 1 h. The membranes were incubated with one of the following primary antibodies overnight at 4°C: rabbit anti-IκBα (1 : 1000), mouse anti-TLR2 (1 : 1000), rabbit anti-collagen III (1 : 500), rabbit anti-collagen IV (1 : 500) or goat anti-CatB (1 : 1000). After washing the membranes with PBS, the membranes were incubated with horseradish peroxidase- (HRP-) labeled anti-rabbit (1 : 1000, GE Healthcare, UK), anti-mouse (1 : 1000, GE Healthcare, UK), or anti-goat (1 : 1000, GE Healthcare, UK) antibodies for 2 h at 24°C, and then the protein bands were detected by an enhanced chemiluminescence detection system (ECK kit, Thermo Scientific, Rockford, IL, USA) using an image analyzer (LAS-4000; Fuji Photo Film, Tokyo, Japan).