After 30 min incubation at 4°C, the cells were washed twice with FACS buffer, and were then incubated with FITC labelled anti-rat IgG (BD Pharmingen) at 10 µg/ml in FACS buffer for 30 minutes at 4°C, washed twice in FACS buffer and then analysed on a FACScalibur™ analyser (Becton Dickinson) running Cellquest™ software.
Briefly, vehicle and Man A treated cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacolylate buffer and were then incubated with osmium tetroxide (OsO4) at 4 °C for 1 h.
After washing for 3 × 10 min in PBS/0.05% Tween 20 buffer, blots were then incubated for another 1 h with an HRP-conjugated IgG secondary antibody (Santa Cruz Biotechnology Inc, 1 : 5000 dilution).
After extensive washing in TBS buffer, the membranes were then incubated with secondary antibody in blocking buffer containing 5% nonfat milk for 1 hr at room temperature.
HepG2 cells and LO2 cells were grown in 96-well plates and fixed with 4% formaldehyde in PBS buffer for 15 min. Cells were blocked with 5% Casien in PBS buffer, and cells were then incubated with recombinant scFv N14 antibody (5 μg/ml) at RT for 2 h.
Cells were lysed in NP-40 lysis buffer, and lysates were then incubated with either GST or GST fusion protein at 4°C for 2 hours, followed by the addition of 20 µl glutathione-Sepharose 4B beads.
After extensive washing with TBST buffer, the blots were then incubated with goat anti-rabbit, horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.
After treating 30 min to 1 hr in Odyssey blocking buffer, the membranes were then incubated for 1 hr at RT with 1 10,000 of anti-PGK using the same IgG-HRP-secondary antibody as described.
For the elastase assay, 400 μL of filtrate was added to a solution of 0.45 % elastin-congo red (w/v) (Sigma-Aldrich) resuspended in buffer B. The samples were then incubated shaking at 37 °C for 2 h.
These beads (∼500 μl slurry per 1 l culture) had been pre-equilibrated in lysis buffer with inhibitors and were then incubated with lysate for 1 hr at 4°C.
