Laemmli sample buffer

After experimental intervention and measurement of ocular parameters, eyeballs were enucleated from C57BL/6 J mice. For protein expression analysis, isolated cornea, lens, retina, choroid and sclerae from eyeballs were homogenized in RIPA buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA, 5 mM benzamidine, 10 mM β-glycerophosphate, 1 mM Na₃VO₄, 50 mM NaF, and 1 mM PMSF) containing Halt protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA). After centrifugation, protein concentration was measured using the BCA (Pierce BCA Protein Assay Kit, Thermoscientific, Waltham, MA, USA) method and adjusted to 1.0 g/L with Laemmli sample buffer (Nacalai Tesque, Kyoto, Japan). Samples were stored at − 30 °C until further use.

After anesthesia as described above, mice were euthanized by cervical dislocation followed by enucleation of eyes for further tissue isolation. Sclera samples were homogenized in RIPA buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM benzamidine, 0.1% sodium deoxycholate 1 mM EDTA, 1 mM Na3VO4, and 1 mM PMSF) containing Halt protease inhibitor cocktail (ThermoFisher Scientific, USA). The protein concentration was measured using a bicinchoninic acid (BCA) protein assay and adjusted with Laemmli sample buffer (Nacalai Tesque). Extracted protein samples were resolved by SDS-PAGE, then transferred to PVDF membranes (Merck Millipore, MA, USA), blocked with Blocking One (Nacalai Tesque, Tokyo, Japan). After that, the membrane was incubated overnight at 4°C with IRE1 alpha, IRE1, phosphor-eIF2α, eIF2α, ATF6 and β-actin antibodies at 4°C. The corresponding secondary antibody (1:10000) was incubated with the membrane at room temperature for 1 h. The SuperSignal West Femto Maximum Substrate (Thermo Fisher Scientific) was used for visualization. SDS-PAGE was performed on 10% acrylamide gels using protein size markers (MagicMark XP Western Protein Standard, Thermo Fisher Scientific).

After experimental intervention and measurement of ocular parameters, eyeballs were enucleated from C57BL/6 J mice. For protein expression analysis, isolated cornea, lens, retina, choroid and sclerae from eyeballs were homogenized in RIPA buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA, 5 mM benzamidine, 10 mM β-glycerophosphate, 1 mM Na₃VO₄, 50 mM NaF, and 1 mM PMSF) containing Halt protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA). After centrifugation, protein concentration was measured using the BCA (Pierce BCA Protein Assay Kit, Thermoscientific, Waltham, MA, USA) method and adjusted to 1.0 g/L with Laemmli sample buffer (Nacalai Tesque, Kyoto, Japan). Samples were stored at − 30 °C until further use.