Anti phospho nf κb

The lung tissues from the experimental rats and lung fibroblasts treated with Chol-HCQ were homogenized in RIPA lysis buffer (Beyotime Biotech, China) containing 1 mM phenylmethylsuphonyl fluoride. The lysates were then centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were collected and stored at −80 °C. A BCA protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine the protein concentrations. Equal amounts of protein were loaded and run on 10% SDS-PAGE gels, transferred onto Millipore PVDF membranes and blocked with 4% BSA. Then, the membranes were incubated with primary antibodies at 4 °C. The following primary antibodies were used: (1) anti-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (2) anti-phospho-NF-κB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (3) anti-ERK1/2 (AbcamPLC, Boston, MA, USA), and (4) anti-phospho-ERK1/2 (Thr202/tyr204) (AbcamPLC, Boston, MA, USA). Antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody and the blots were developed with the ECL-Plus reagent (Millipore, MA, USA). The blots were tested for GAPDH (AbcamPLC, Boston, MA, USA) to confirm equal protein loading.

Protein lysates from the MAC-T cells were prepared with ice-cold PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) in accordance with the manufacturer's instructions. Lysates were precipitated at 1200 rpm for 10 min at 4°C. The total protein content was determined using the Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein extracts in lysis buffer were separated using 4%–12% polyacrylamide gels (German, Sigma-Aldrich) and then transferred onto a nitrocellulose membrane. Resolved proteins were blotted onto PVDF transfer membranes (Millipore Co., Bedford, MA) and then blocked with 10% nonfat milk in TBST. The membranes were incubated with anti-CYP1A1 (Sangon Biotech, Shanghai, China), anti-NF-κB (Bioss, Beijing, China), anti-phospho-NF-κB (Santa Cruz Biotechnology lnc., Santa Cruz, CA, USA), and anti-GAPDH (TransGene) antibodies at 4°C overnight. The membranes were washed three times with TBST for 5 min before incubation with HRP-conjugated secondary antibodies. Finally, the immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit (Beyotime).

Cells were seeded at a density of 3.0 × 10⁵ cells/well in a 4-well chamber slide. After 24 h, the media were changed to serum-free media, containing the 100 ng/mL of BV. After 1 h, the cells were treated with 100 ng/mL of PgLPS (InvivoGen) for 7 h. The treated-cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were treated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 2 min to permeabilize. Following permeabilization, the cells were blocked in PBS, containing 5% bovine serum albumin, at room temperature for 1 h. After blocking, the cells were incubated with diluted primary antibody overnight at 4 °C, and secondary antibody was performed at room temperature for 4 h. The nuclei were stained with Hoechst 33342 solution for 20 min. Slides were mounted by using fluorescence mounting medium (Dako, Santa Clara, CA, USA). Specimens were examined and photographed by the using confocal microscope system (Nikon, Tokyo, Japan). Antibodies used in the present study were the following: anti-TNF-α; anti-phospho-NF-κB (Santa Cruz); anti-mouse-, and anti-rabbit-biotinylated secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Thermo).