Salicylate sodium salicylate

Pyridoxamine-dihydrochloride-monohydrate (4-aminomethyl-3-hydroxy-2-methyl-5-oxymethylpyridinedihydrochloride) was purchased from Tokyo Chemical Industry Co., Japan. The pyridoxamine dihydrochloride was given at 1 g/L (~200 mg/kg/day) in the drinking water from 4 to 20 weeks of age for 16 weeks, resulting in the serum concentration at 0.42 μM (<0.03 μM in nontreated mice). Salicylate (sodium salicylate, Sigma-Aldrich, Japan) was given at 3 g/kg in their diets, yielding the serum concentration at 102.6 μM (not detected in nontreated mice). For the insulin treatment group, a long-acting insulin analog of insulin detemir (Levemir, Novo Nordisk) was subcutaneously injected twice a day at the dose of 2 units/kg body weight.
The following five animal groups were subjected to phenotypic analyses: (1) nondiabetic control (n = 8), (2) diabetic mice without treatment (n = 19), (3) diabetic mice with insulin treatment (n = 8), (4) diabetic mice with salicylate treatment (n = 12), and (5) diabetic mice with pyridoxamine treatment (n = 15).

A rabbit polyclonal antibody against DDDDK-tag was used as the anti-Flag antibody (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan). Mouse monoclonal antibodies against α-tubulin (DM1A (epitope; 426–450 residues), NeoMarkers, Fremont, CA, USA), Cy3-conjugated-β-tubulin (both antibodies used for general tubulin staining), and acetylated-α-tubulin (clone 6-11B-1) (Sigma, St. Louis, MO, USA), were used. Human anti-centromere antibody (Antibodies Inc., Davis, CA, USA) was also used. DAPI (4,6-diamidino-2-phenylindole dihydrochloride hydrate) (Sigma), and tubacin (Enzo Life Sciences, Farmingdale, NY, USA) were used. Salicylate (sodium salicylate) was purchased from Sigma. NAP peptide (Biorbit, San Francisco, CA, USA) was aliquoted and stocked as a 1 mM solution in 75% dimethyl sulfoxide at −20 °C.