2.11. Mn2+-Phos-tag™ SDS-PAGE Analysis

Phos-tag acrylamide (NARD, Hyogo, Japan) at a concentration of 5 mM and supplemented with 10 mM MnCl₂ was added to the SDS-PAGE gels. Prior to blotting, the gels were first soaked in a transfer buffer containing 8 mM EDTA for 15 min to remove MnCl₂, and the duration of blotting was extended to 3 h [36]. The VDAC1 (Sigma, USA) antibody at a 1 : 1000 dilution was then added to determine the extent of VDAC1 phosphorylation via Phos-tag gel blotting.