Optimization of α‐Synuclein and Tau Detection by Immunoblot in Enteroendocrine Cell Lines

ABSTRACT Background Enteroendocrine cells (EECs) are dispersed along the intestinal mucosa and transduce luminal stimuli into hormonal signals. EECs exhibit neuron‐like features and express both α‐synuclein and tau, two proteins pathologically and genetically linked to Parkinson disease (PD). These observations support the hypothesis that EECs may be involved in disease development in the “body‐first” PD subtype. Cellular models represent invaluable tools for studying the role of α‐synuclein and tau in PD pathogenesis. However, the sensitivity and specificity of commercial antibodies for detecting α‐synuclein and tau in EEC cell lines remain unclear. Methods We tested by immunoblot a panel of commercial total‐α‐synuclein and total‐tau antibodies on protein lysates from three EEC cell lines: GLUTag, NCI‐H716, and STC‐1. Pharmacological and biochemical manipulations were applied to assess the specificity of antibodies against phosphorylated α‐synuclein and tau. Key Results Five antibodies detected total α‐synuclein in NCI‐H716 lysates, whereas the antibodies D1M9X and Tau12 detected total tau in GLUTag and NCI‐H716 lysates, respectively. α‐synuclein and tau protein levels were comparable between naïve and differentiated NCI‐H716 cells. Four phospho‐specific antibodies revealed phospho‐α‐synuclein S129 in NCI‐H716. Of the nine phospho‐tau antibodies tested, six recognized tau phosphorylated at specific epitopes (T181, S199, T231, S356, S396, and S404) in GLUTag cells. Conclusions and Inferences Our results indicate that NCI‐H716 cells represent an ideal EEC model for studying α‐synuclein expression and phosphorylation, whereas GLUTag cells are preferable for investigating tau protein biology. This work provides a comprehensive antibody toolbox to dissect the physiological and pathological role of α‐synuclein and tau in EECs.