Imagequant las 4000 mini

Lung tissues were homogenized in RIPA lysis buffer containing phosphatase inhibitors (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Two milligrams of each lysate were applied to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond-P membranes (GE Healthcare Ltd, Buckinghamshire, UK). After blocking with Blocking One-P (Nacalai Tesque Inc, Kyoto, Japan), the membranes were incubated with anti-AQP5 (GTX11586, x2000, GeneTex, Inc., Irvine, CA, USA) or anti-β-actin (PM053, 1:1000, MBL Co., Nagoya, Japan). They were washed and incubated with peroxidase-conjugated anti-rabbit IgG (1:5000, MBL Co.). The protein bands were visualized with Chemi-Lumi reagents (Nacalai Tesque Inc) and image-captured with a CCD camera system, ImageQuant LAS 4000 mini (GE Healthcare Ltd).

The M0 THP-1 macrophages were treated with 5% QPD or 5% XBD for 24 h. Then the cells were stimulated with or without 1 µg/ml LPS and 20 ng/ml IFN-γ. The total intracellular protein was collected at 1 h and 2 h post-stimulation. IκBα, phospho-IκBα, NF-κB p65, and phospho-NF-κB p65 protein levels were assessed by western blot using IκBα (L35A5) mouse mAb, phospho-IκBα (Ser32/36)(5A5) mouse mAb, NF-κB p65 (D14E12) XP^® rabbit mAb, and phospho-NF-kB p65 (Ser536) (93H1) rabbit mAb (Cell Signaling Technology, United States), respectively. Secondary antibodies were HRP-labeled goat anti-mouse or anti-rabbit IgG (Proteintech, China). The protein bands were visualized using an ECL chemiluminescent detection kit (Millipore, United States) in an ImageQuant LAS 4000mini (GE, United States).

One or two-days post infection, cells were collected and washed in PBS. Cells were resuspended in HED buffer (20mM HEPES pH 7.4, 5mM EDTA, 1mM DTT, 10% glycerol) supplemented with cOmplete Protease Inhibitor Cocktail (Roche). Cells were then submitted to one freeze/thaw cycle and sonicated 15 cycles of 30 sec ON / 30 sec OFF using Bioruptor Pico device (Diagenode) at 4°C. Cell lysates were spun down at 14,000 rpm for 15 minutes to remove cell debris. Proteins were quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Forty μg of proteins were incubated overnight at 37°C with 1 pmol of a fluorescent oligo substrate (5’-ATTATTATTATTCAAATGGATTTATTTATTTATTTATTTATTT-Cy5-3’), 1mM ZnCl₂, 0.025U uracil DNA glycosylase (NEB), 2 μL 10x UDG buffer (NEB) and 100 μg/mL RNAse A (Thermofisher Scientific). Reaction mixture was treated with 50 mM NaOH and heated to 95°C for 10 minutes to cleave DNA probes at the abasic site. Reaction mixture was then neutralized with 50 mM HCl and mixed with 1.25x formamide buffer. Substrates (43 bases-long) were separated from products (30 bases-long) on a 15% tris-borate-EDTA (TBE)-urea gel. The Cy5-labeled substrates and deamination products were detected using ImageQuant LAS4000 mini (GE Healthcare Life Sciences). Densitometry analysis was done using ImageJ software [51 (link)].

Protein extracts from cells were prepared using a lysis buffer (200 mM Tris-HCl [pH 7.5], 1.5 M NaCl, 10 mM EDTA, 10 mM EGTA, 25 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1 mM Na3VO4, 50 mM NaF) supplied with a protease inhibitor cocktail (Roche Diagnostics). Protein samples of 20 ug each sample were separated on a 10% polyacrylamide gel and analyzed by Western blot using anti-HA (Proteintech, 51064-2-AP, 1:3,000) and anti-Gapdh (Proteintech, 10494-1-AP, 1:1,000) antibodies. Peroxidase-conjugated rabbit immunoglobulin G (IgG; Jackson ImmunoResearch, 1:2,500) was used as the secondary antibody. Western blots were developed using ImmobilonTM Western Chemiluminescent HRP Substrate (Merck Millipore), and analysis was performed with a Luminescent Image Analyzer (GE, ImageQuant LAS 4000 mini). The results were quantified by using the ImageJ software.

CAB cells (or EPC cells) were seeded in 10 cm² dishes overnight and then transfected with a total of 10 μg of various plasmid combinations. The transfected cells were washed twice with 10 mL ice-cold PBS and then lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatant was transferred to a 1.5 mL clean tube and incubated with 25 μL anti-Flag Affinity gel (Sigma-Aldrich) or anti-HA Magnetic Beads (Thermo Fisher) overnight at 4°C with constant rotating incubation. Immunoprecipitated proteins were collected by Magnetic Stand (Promega), washed five times with lysis buffer, and resuspended in 100 μL SDS-PAGE protein loading buffer (Beyotime). The immunoprecipitates and whole cell lysates (WCLs) were separated by 10–12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore) for subsequent western blot analysis. Antibodies were diluted as follows: anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA antibody (Cell Signaling Technology) at 1:3,000, anti-Myc antibody (Abcam) at 1:2,000, and HRP-conjugated anti-rabbit IgG (Thermo Scientific) at 1:5,000. Images were captured by ImageQuant LAS 4000mini (GE). Results were representative of three independent experiments.

For Western blotting, cells were collected at indicated time points and then were lysed by RIPA lysis buffer (Beyotime, Nantong, China). Cell lysates (90 μl) were mixed with 30 μl SDS loading buffer and boiled for 5 min at 100°C for SDS/PAGE. Primary antibodies were diluted according to instructions. HRP-labeled secondary antibody was used at a dilution of 1:3000. Immuno-reactive bands were revealed by enhanced chemiluminescence (Clarity™ Western ECL Substrate, Bio-Rad) and visualized by the Image Quant LAS 4000 mini (GE). Band intensities were quantified and analyzed with ImageJ and normalized against the level of β-actin.