Phospho-antibody microarray analyses for islets of control and Mettl14 knock-out mice

β-cell specific Mettl14 knock-out mice display reduced N6-methyladenosine (m6A) levels and recapitulate human Type II diabetes (T2D) islet phenotype with early diabetes onset and mortality secondary to decreased β-cell proliferation and insulin degranulation. To gain insights into the role of m6A in regulating the IGF1/insulin -> AKT - > PDX1 pathway and to dissect the signaling networks modulating AKT phosphorylation, we subjected freshly isolated islets from control and Mettl14 knock-out mice to phospho-antibody microarrays. Mettl14fl/fl mice on a C57BL/6N background was created and provided by Dr. Chuan He (University of Chicago). To obtain β-cell specific Mettl14 knock-out mice, Mettl14fl/fl mice were breed with B6(Cg)-Ins1tm1.1(cre)Thor/J (Jackson Labs, USA) and male mice wild-type for nicotinamide nucleotide transhydrogenase (Nnt) mutation were used. Mice were weaned at 3 weeks of age and maintained on a chow diet (PicoLab® mouse diet 20 – 5058). Islet isolations were performed in anesthetized mice and their pancreas infused with liberase (Roche, Germany). Following incubation at 37C for 17 min the digested pancreases were washed filtered through a 400µm filter and run on a Histopaque (Sigma, USA) gradient. The purified islets were handpicked, counted and cultured overnight in 7mM glucose RPMI media (Gibco, USA) containing 10% FBS and 1% PS (Gibco, USA). Islets were handpicked, washed 2 times with ice-cold DPBS by self-sedimentation. Protein lysates from 200 size-matched islets from each sample (4 control pools and 2 Mettl14 knock-out pools) were used for Kinex™ KAM-1325 antibody microarray analyses.