Luminescent image analyzer

Protein samples were separated on a 10% SDS-polyacrylamide gel and then transferred to nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Membrane was blocked with 5% skimmed milk in Tris-buffered saline with Tween (TBST) (25 mM Tris, 200 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for 1 h at room temperature before incubation with primary antibody (goat anti-human PIGR antibody [R&D Systems 1:5000] or mouse anti-human α-tubulin antibody [Sigma-Aldrich 1:500]) overnight at 4 °C. After washing three times with TBST, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody (rabbit anti-goat IgG HRP-conjugated antibody [R&D Systems 1:1000] or goat anti-mouse IgG HRP-conjugated antibody [Sigma-Aldrich 1:70,000]) for 1 h at room temperature. The membrane was washed thrice with TBST and enhanced chemiluminescent detection was performed using Immobilon Western chemiluminescent HRP substrate (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The chemiluminescent signal was visualized using a Luminescent Image Analyzer (Fujifilm Life Science, Cambridge, MA, USA).

The peptides, 13 aminoacids long, were synthesized according to standard solid phase synthesis protocols [44] (link), using an automatic SPOT synthesizer (Intavis, Koeln, Germany). In this approach, peptides are synthesized in array format, bound to cellulose membranes, which are activated with amino PEG (Intavis). The chemistry uses Fmoc protected amino-acids, with protected side chains, which are deprotected at the end of the synthesis.
Before use, the membranes were wet in ethanol and washed repeatedly in PBS. Membranes were blocked overnight at 4°C in buffer HT containing 5% BSA (blocking buffer).
The binding assay was performed incubating the membranes with 0.2 µM GST-fused GF14 isoforms, or GST alone as a negative control, in blocking buffer overnight at 4°C. After washing membranes three times for 10 minutes with HT buffer, the anti-GST peroxidase-conjugated antibody was added 1/1000 in blocking buffer and incubated for 2 h. Membranes were washed three times with HT buffer, and quantification of peptide bound GF14 proteins was carried out using a chemo-luminescence substrate and the LAS-3000 instrument (Luminescent Image Analyzer, Fujifilm).
Densitometric analysis of positive spots was performed using the ImageJ image processing program [41] and data expressed as Integrated Densitometric Value (the product of the area and mean gray value).

AGS cells (6 × 10⁵) or GES-1 cells (4 × 10⁵) were seeded on 6-well plates for 16 h and infected with H. pylori (MOI 100) for 6 h. The infected cells were washed five times with DPBS, and whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (Sigma-Aldrich) with protease inhibitor cocktail (Calbiochem) and phosphatase inhibitor cocktail (Calbiochem). The samples were then analyzed by SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane (GE Healthcare). Each membrane was blocked with 5% (w/v) dry milk in Tris-buffered saline (50 mM Tris, 150 mM NaCl, 1 mM CaCl₂, pH 7.4) containing 0.01% (v/v) Tween-20 at room temperature for 1 h and incubated at 4 °C for 16 h with rabbit monoclonal anti-LC3B (Sigma; 1:4000), mouse anti-β-actin (Sigma; 1:5000), rabbit anti-Cathepsin D (Abcam; 1:5000), rabbit anti-Lamp-1 (Abcam; 1:1000) or rabbit anti-GAPDH (Abcam; 1:5000). Each blot was washed three times and incubated at room temperature for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology; 1:5000). The blots were visualized with ECL western blotting detection reagents (Millipore; WBKLS0500) and visualized with a Luminescent Image Analyzer (LAS4000, Fujifilm).

Telomere terminal restriction fragments were measured as previously described [2 (link)]. Briefly, telomere length was measured by Southern blotting. Two μg of digested DNA was separated on 0.7% agarose gel. Hybridization was carried out with 3′-end DIG-labeled d(TTAGGG)₄ (Roche Molecular Biochemicals, Mannheim, Germany) and detected as recommended by the manufacturer. The resulting X-ray film was scanned with a luminescent image analyzer (Fujifilm, Tokyo, Japan), and the telomere signals in each lane were quantified as a grid object, defined as a single column with 25 rows, using Image Gauge Software 2.54 (Fujifilm).