Western Blot Analysis of Cytochrome b5

Microsomes containing 30 μg microsomal proteins were subjected to electrophoresis on the 15 % polyacrylamide gel and applied onto a polyvinylidene fluoride (PVDF) membrane as reported [44 (link)]. The membranes were then exposed to specific rabbit polyclonal anti-cytochrome b₅ (1:750, AbCam, MA, USA) antibodies overnight at 4 °C and the antigen–antibody complex was visualized with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:1428, Sigma-Aldrich, USA) and 5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium as chromogenic substrate. Protein bands were expressed as arbitrary units (AU)/mg protein as described previously [14 (link), 19 , 20 (link)]. Antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) (1:750, Millipore, MA, USA) was used as loading control as recommended by the antibody producer.