Anti ccr5

The cells were taken from the incubator to remove the culture medium and washed twice with precooled PBS. Proteins were harvested using whole cell lysis assay (KeyGEN Biotech, China) according to the manufacturer's instructions. After valuing the concentration and equaled the quantity of loading samples, the proteins of different groups were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% nonfat milk in TBST at room temperature for 1 h, followed by incubation with antibodies against targeting protein overnight at 4°C. The primary antibodies including anti-CCR5, anti-TRAF6, anti-NF-kB, and anti-PI3K were purchased from Abcam (Cambridge Science Park, UK), and used as the following concentration: anti-CCR5: 1 : 1000; anti-TRAF6:1 : 2000; anti-NF-kB:1 : 500; anti-PI3K:1 : 250; and anti-β-Actin:1 : 5000. Following being washed with TBST buffer, the membrane was stripped with appropriate HRP-conjugated secondary antibodies (Abcam, CA, USA) for 1 h at room temperature, followed by visualized using the enhanced Western Bright ECL reagents (Cell Signaling Technology, USA). Eventually, bands were imaged and analyzed by a chemiluminescence detection system (Bio-Rad, USA). The experiments were repeated at least three times to ensure reproducibility.

Cells were cultured or treated by rBFT1, followed by harvesting at predetermined time points. After extensive washing, cells were lysed using Whole Cell Lysis Assay (KeyGEN Biotech, China) according to the manufacturer’s instructions and proteins from the lyste were harvested by centrifugation at 12,000 × g for 5 min at 4°C. Protein concentrations were determined by the BCA Assay kit (KeyGEN Biotech, China). Total protein (42 kDa) from each lysate was then separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by incubation with antibodies against protein of interest overnight at 4°C. The antibodies used in this study included anti-NF-κB/P65 (1:100 in PBS), anti-Macrophage Inflammatory Protein 1α/CCL3 (100 μg/mL in PBS), anti-CCR5 (1:100 in PBS), anti-PI 3 Kinase/P85α (1:200 in PBS), and anti-TRAF6 (1 µg/mL in PBS) (Abcam, CA, USA). After washing with TBST, membranes were striped with appropriate HRP-conjugated secondary antibodies (1:5,000, Abcam, CA, USA) for 1 h at room temperature, followed by visualization using the enhanced Western Bright ECL reagents (Cell Signaling Technology, USA). Protein bands were scanned and analyzed by a chemiluminescence detection system (Bio-Rad, USA).