Coomassie plus

Protein samples were obtained from 15 confluent plates of non-synchronized animals, washed using M9 buffer and lysed in RIPA buffer, followed by three sonication cycles of 30 seconds on ice. Protein levels were quantified using Bradford reagent (Coomassie Plus, Thermo Scientific, #23238) and 20 µg was loaded on a 10% polyacrylamide gel, together with molecular weight standards (PageRuler, ThermoScientific, #11852124). Antibodies used include rabbit anti-GFP (1:1000, Sigma Aldrich, #G1544), mouse anti-Tubulin (1:1000, Cell Signaling, #3873) and secondary HRP-linked antibodies directed against mouse IgG (1:10,000, Cell Signaling, #7076S) and rabbit lgG (1:10,000, Cell Signaling, #7074S). HRP activity was revealed using ECL (Supersignal, West Atto Ultimate, ThermoScientific, #A38554). Anti-GFP was used to monitor expression of LGG-1 in the MAH215 and MTM38 strains.

The APPΔC31 ELISA developed in partnership with Enzo Life Sciences (ADI-900-227) follows a standard sandwich ELISA design wherein a monoclonal N-terminal APP-specific antibody is used to coat the wells of the microtiter plate (capture antibody) and the anti-APPΔC31 cleavage site-specific polyclonal antibody serves as the detection antibody. An HRP-conjugated secondary antibody is then added and a colorimetric signal generated by enzymatic conversion of the tetramethylbenzidine (TMB) substrate. Protein levels were first determined using a colorimetric coomassie assay (Coomassie Plus, Thermo).

Brain progranulin protein levels were analyzed with an ELISA kit (Adipogen) using the manufacturer’s protocol. Hippocampus or frontal cortex samples were homogenized in lysis buffer (10 mm Tris, pH 7.5, 10 mm NaCl, 3mm MgCl₂, 1mm EDTA, and 0.05% NP-40) with protease inhibitors (Halt protease inhibitor cocktail, Thermo Scientific) and protein concentration was determined by Bradford assay (Coomassie Plus, Thermo Scientific). The samples were diluted to 1 mg protein/ml with lysis buffer, and then diluted 1:1 with ELISA sample buffer. Samples were run in duplicate, with 50 µg of protein loaded per well. The progranulin content was calculated using a standard curve of recombinant progranulin from the same plate, and was normalized to the amount of protein loaded per well.

Ileal samples were homogenized using ice-cold T-Per^® tissue protein extraction reagent (Thermo Fischer Scientific, Inc., Waltham, MA, USA) containing 1 mM phenylmethylsulphonyl fluoride (Thermo Fisher Scientific, Inc.) and Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.), and the homogenate was centrifuged at 10,000× g for 10 min. Protein concentration in collected supernatant was quantified using the Coomassie Plus (Bradford) assay kit (Thermo Fisher Scientific, Inc.). Protein expression levels of IL-1β, IL-8, IL-10, and IL-18 in ileal tissue homogenates were determined using a commercially available chicken ELISA kit (LifeSpan BioSciences, Inc., Seattle, WA, USA) according to the manufacturer’s instructions. Before the assay, original ileum homogenates were diluted with the kit’s sample diluent 1:26 for IL-8 and IL-10, 1:121 for IL-1β, and 1:961 for IL-18. The optical density was determined using plate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA) set to 450 nm. Results were calculated from standard curve using SpectraMax M2 software.