Blocking reagent

Nitrocellulose membranes were spotted with 5 µL of each coating mAb (0.45 mg/mL in PBS). To block additional protein-binding sites, the membrane was incubated overnight at 4° C with PBST containing 5 % blocking reagent (BioRad) with shaking. The membrane was cut to separate the original mAb spots. Each of the resulting nitrocellulose discs were then separated in one of 12 wells of a cell culture plate (Costar). Each well was filled with 500 µL of the specific polysaccharide diluted to 25 µg/mL in PBST/3 % blocking reagent. The plate was incubated for 2 h at RT with mild shaking. The specific biotinylated monoclonal antibody diluted 1:100 in 500 µL PBST/3 % blocking reagent, was added to each well followed by incubation at RT for 1 h with shaking. After extensive washing, the plate was incubated for 45 min with 500 µL of horseradish peroxidase-conjugated streptavidin (Thermo Scientific) diluted 1:5000 in PBST/3 % blocking reagent. The blot was developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) following manufacturer instructions.

Protein lysates were obtained by homogenizing the pancreatic and brain tissues (whole brain tissue) with T-PER (Tissue-protein extraction lysis solution) (50 mg/500 μl lysing solution) along with 10 μl protease inhibitor cocktail and 10 μl phosphatase inhibitor. Homogenization was done under ice and centrifuged at 10,000×g for 5 min to collect the debris. Protein quantification was done by Bradford assay and 40 μg of protein was loaded into the SDS-PAGE. The separated protein samples were transferred to the nitrocellulose membrane by using Bio-Rad Transblot turbo transfer system. After transfer, membrane was blocked by using 5% Bio-Rad blocking reagent. Blocked membrane was incubated with 1:1000 dilution primary rabbit antibodies (Cell Signaling Technology, USA) for 12 h at 4 °C. After treatment, the membrane washed and incubated with secondary antibody (1: 20,000) treatment for an hour. Bands were observed in the chemiluminescence document reader, after addition of Immobilon Western chemiluminescent HRP substrate at standardized exposure time. Bands of the two groups were analyzed by densitometry analysis compared to β-Actin (Major Science image analysis software).

Protein samples were resolved on 4% to 20% Mini-Protean TGX precast protein SDS-PAGE gels (Bio-Rad Laboratories; Hercules, CA) and blotted on polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were incubated in TBS buffer containing 0.1% (vol/vol) Tween 20 (TTBS) and 5% (wt/vol) blocking reagent (Bio-Rad) with gentle shaking at room temperature for 30 min. Primary antibodies were added as described above with mouse anti-strep (Millipore Sigma no. 71590-M, 1:1,000 dilution) and mouse anti-His antibodies (Santa Cruz Biotechnology, Dallas, TX; no. SC-53073, 1:400 dilution) diluted in TTBS with 0.5% (wt/vol) blocking reagent, followed by incubation with gentle shaking at room temperature for 12 to 14 h. Membranes were washed three times with TTBS, followed by incubation (2 h at room temperature with gentle shaking) with secondary antibodies (goat anti-rabbit-HRP [Thermo Fisher Scientific no. 65-6120, diluted 1:3,000] and donkey anti-mouse-Alexa 647 [Thermo Fisher Scientific no. A-31571, diluted 1:1,000]) diluted in TTBS with 0.5% (vol/vol) blocking reagent. Membranes were washed twice in TTBS and once in TBS before Western blot detection. Blots were visualized using the Bio-Rad ChemiDoc MP imaging system.