2.8. Protein expression analysis by Western blot

Liver samples (approximately 6 mg) were homogenized (6,500 rpm, 30 s, 4°C, Precellys 24 tissue homogenizer) in RIPA buffer (450 μl) containing protease inhibitor cocktail and centrifuged (4 min, 4°C, 16,000× g). Protein concentration was quantified by using a bicinchoninic acid protein assay and samples were prepared (5 min at 95°C) in Laemmli solubilization buffer (60 mmol·L⁻¹ Tris–HCl, 10% glycerol, 0.01% bromophenol blue, 2% sodium dodecyl sulfate, pH 6.8, 5% β‐mercaptoethanol). The protein extract (20 μg) was separated by 14% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked (1 h, 25°C) in TBS‐T (5% defatted milk, 20 mmol·L⁻¹ Tris buffered saline with 0.1% Tween‐20). All antibody dilutions and incubations were performed in TBS‐T. 11β‐HSD1 protein expression was measured with rabbit polyclonal anti‐11β‐HSD1 antibody (1:1,000, 4°C, overnight). The membrane was washed and incubated with HRP‐conjugated goat anti‐rabbit secondary antibody (1:2,000, 25°C, 1 h). H6PD protein expression was determined using rabbit polyclonal anti‐H6PD antibody (1:1,000, 4°C, overnight) and HRP‐conjugated goat anti‐rabbit secondary antibody (1:2,000, 25°C, 1 h). ACTB was detected using mouse monoclonal anti‐ACTB antibody (1:1,000, 4°C, overnight) followed by HRP‐conjugated goat anti‐mouse secondary antibody (1:4,000, 25°C, 1 h). Protein content was visualized by Immobilon Western Chemiluminescence HRP substrate kit. 11β‐HSD1 and H6PD were quantified by densitometry normalized to ACTB protein levels using ImageJ software (version 1.53n, RRID:SCR_003070). The Immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., ²⁰¹⁸).