Densitometric software

Whole fresh retinas were rapidly isolated and homogenized in ice-cold lysis buffer: 150 mM NaCl, 20 mM Tris, pH 8.0, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA, supplemented with 2 mM NaVO3, and protease and phosphatase inhibitors. Protein samples were resolved on SDS polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Life Science, Mississauga, ON). Blots were incubated with each of the following antibodies: pAMPK^Thr172 (0.027 μg/ml, Sigma), total AMPK (1:1000, Sigma), phospho-LKB1 (Ser²⁴⁸, 0.183 μg/ml, Sigma), total LKB1 (0.024 μg/ml, Sigma), or β-actin (0.5 μg/ml, Sigma-Aldrich), followed by anti-rabbit or anti-mouse peroxidase-linked secondary antibodies (0.5 μg/ml, GE Healthcare, Mississauga, ON). Blots were developed with a chemiluminescence reagent (ECL, Amersham Biosciences) followed by exposure of blots to ChemiDoc MP System (Bio-Rad Life Science). Analysis was performed using densitometric software (Bio-Rad Life Science) and three independent western blots were carried out using retinal samples from distinct experimental groups.

Western blot analysis was performed as described previously [45 (link)]. In brief, cells were lysed with lysis buffer (Beyotime, Shanghai, China) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 14,000 × g for 10 min at 4°C, the extracted proteins were resuspended, and the concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime). Equal amounts (30 µg) of protein were resolved by SDS-PAGE and electro-transferred onto the PVDF membrane (Millipore). The immunoblots were then blocked with 5% fat-free milk for 1 h, and further incubated overnight with primary antibodies against ZFP36L1 (1:1000 dilution, ab42473, Abcam, USA) and GAPDH (1:3000 dilution, ab181602, Abcam, USA) at 4°C. After incubation with peroxidase-conjugated secondary antibodies for 1 h, the blots were washed and then visualized using an electrochemiluminescence (ECL) detection kit (ThermoBiotech Inc, Rockford, IL, USA). The chemiluminescent signal on the blots was determined and quantified using densitometric software (Bio-Rad, California, USA).