Quantifying Amidated Chromogranin A

To mimic the effect of gene silencing, cells were cultured for 24 h with the irreversible PAM inhibitor 4-phenyl 3-butenoic acid (4P3BA; Sigma) at a concentration of 500 μM67 (link). Whole-cell extracts (WCE) from siRNA or inhibitor-treated EndoC-βH1 cells were prepared as previously described68 (link). Supernatant was harvested directly from cells and filtered with a 0.22μm filter. Protein yields were determined by the Bio-Rad Protein Assay according to manufacturer’s instructions. For western blotting, 10μg WCE and 0.5% total supernatant volume were analysied via denaturing SDS-PAGE. Antibodies specific for CgA-Gly (Abcam, ab52983)45 and total CgA (Abcam, ab15160) were used to calculate the ratio of amidated to non-amidated CgA using Image Lab 5.0.

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Thomsen S.K., Raimondo A., Hastoy B., Sengupta S., Dai X.Q., Bautista A., Censin J., Payne A.J., Umapathysivam M.M., Spigelman A.F., Barrett A., Groves C.J., Beer N.L., Manning Fox J.E., McCarthy M.I., Clark A., Mahajan A., Rorsman P., MacDonald P.E, & Gloyn A.L. (2018). Type 2 Diabetes Risk Alleles in PAM Impact Insulin Release from Human Pancreatic Beta Cells. Nature genetics, 50(8), 1122-1131.

Publication 2018

Protein Assay ab15160

Antibodies Assay Cell extracts Cells Phenyl acid Protein Rad protein Sds page Sirna

Corresponding Organization :

Other organizations : Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, University of Alberta, Health Data Research UK, Wellcome Centre for Human Genetics, Churchill Hospital

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Variable analysis

independent variables

Irreversible PAM inhibitor 4-phenyl 3-butenoic acid (4P3BA) at a concentration of 500 μM

dependent variables

Ratio of amidated to non-amidated CgA

control variables

Cell line (EndoC-βH1 cells)
Duration of treatment (24 h)

controls

Positive control: siRNA-treated EndoC-βH1 cells
Negative control: Not explicitly mentioned

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