Antibody against β actin or gapdh

Expressions of proteins were quantified by western blot analysis as described previously (33 (link)–35 (link)). Whole tissue proteins were extracted from a segment of large intestine by using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethane sulfonyl fluoride (PMSF) (Sigma-Aldrich), and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Protein concentrations were determined by using the bicinchoninic acid assay (BCA, ThermoFisher). Protein samples were resolved on NuPAGE 4-12% Bis-Tris gels (Life technologies), and immunoblot analyses were performed using antibodies against phosphorylated α-syn at serine 129 (1:2000; Abcam) or α-syn (1:2000; Abcam). An antibody against β-actin or GAPDH (1:5000; Cell Signaling Technology) was included to monitor loading errors. The intensity of the western blot signals was quantitated using ImageJ software (NIH, Bethesda, MD, USA), and the densitometry analyses are presented as the ratio of protein/β-actin protein, and are compared with controls and normalized to 1.