Semi dry apparatus

Protein concentration of cell lysates was assessed (BCA method); 30 or 50 μg/ml protein samples were added to sample buffer and electrophoresed in SDS-PAGE system, 12% or 15% gel densities. A semi-dry apparatus (GE HealthCare) was used for transference to nitrocellulose membranes (GE HealthCare). Membranes were incubated in 5% non-fat dry milk for 2 h, washed and incubated with primary antibodies at 4 °C overnight, followed by fluorophore-conjugated secondary antibodies. Bands were detected by Odyssey System (LI-COR Biosciences) and quantified by densitometry using the system program. Uncropped western blots are presented as supplemental material (Supplementary Figs S8–S10).

Samples (1/20 vol of fractions containing proteins extracted from the small intestinal tissue) were resolved by AU-PAGE at 150 V and transferred to 0.1-μm nitrocellulose membrane (GE Healthcare) using a semidry apparatus (ADVANTEC) at 2.5 mA/cm² for 30 min. Chemically synthesized ox and rCrp1 prepared as described (44 (link)) were used as standards. The membranes were blocked by BSA-free StabilGuard Immunoassay Stabilizer (Surmodics) at room temperature for 1 h, incubated with biotinylated anti-Crp1 antibody (1 μg/ml, 76-R29, self-produced), which reacts to both ox and rCrp1–4 and 6 (Fig S2) at 4°C overnight. Membranes were incubated with HRP-conjugated streptavidin (GE Healthcare) at room temperature for 1 h. After incubation, the blots were visualized by Chemi-Lumi One Ultra.