Ab231094

Total proteins were extracted from cells and tissues by RIPA buffer containing 1% protease suppressors and phosphatase suppressors. A BCA assay kit (Solarbio, Beijing, China) was used to detect the protein concentration. Total proteins were isolated by SDS‒PAGE, and the separated proteins were transferred to PVDF membranes and blocked with 5% skim milk powder at room temperature for 1.5 h. Then, the following diluted primary antibodies were added: FBN1 (1:1000, ab231094, Abcam, UK), TGF-β (1:1000, ab215715, Abcam, UK), Bax (1: 1,000, ab32503, Abcam, UK), Bcl-2 (1 ab32124, Abcam, UK), cleaved caspase-3 (1:500, ab32042, Abcam, UK), and β-actin (1:1000, ab8226, Abcam, UK) overnight at 4 °C. Next, the membrane was incubated with secondary antibody (1:4000, ab97051, Abcam, UK) for 1 h at room temperature and developed with an ECL kit. Ultimately, the bands were semiquantitatively analyzed by ImageJ software.

Pooled aqueous humor from WT and Tsk mice (n = 10–12 mice/group; 5 independent experiments) were used for immunoblotting. For immunoblotting, 10-30 µl of sample was heated for 5 min at 95 °C with reduced 6× Laemmli SDS sample buffer (Bioland Scientific LLC) and run in SurePAGE, 4–12% Bis–Tris gel (GeneScript). Immunoblot samples were loaded in equal protein concentrations for SDS-PAGE based on Bradford assays. The proteins were transferred onto PVDF membranes (Bio-Rad) and stained with Ponceau S staining solution (Bio-Rad) to verify transfer efficiency. Membranes were washed in Tris-buffered saline plus 0.1% Tween 20 (TBST) and blocked using 5% nonfat dry milk in TBST for 1 h at RT then incubated with primary antibodies (fibrillin-1 (ab231094, Abcam) or TGFβ2 (ab36495, Abcam)) overnight at 4 °C. After washing 3 times, membranes were incubated for 1 h at RT with HRP-conjugated secondary antibody (ab6789 or ab6721, Abcam) in blocking solution. Washed membranes were then incubated with a SuperSignal substrate (Thermo Scientific) for 1 min at RT and signal detection and densitometry analysis were performed with BioRad ChemiDoc XRS system.

The bicinchoninic acid protein assay kit (Thermo Fisher Scientific) was adopted for total protein extraction, followed by measurement of protein concentration. Next, 30‐μg portions of total protein were separated by polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene fluoride membranes (Amersham Healthcare, Chicago, IL, USA). The membrane was then blocked by 5% nonfat milk at room temperature for 1 h, followed by overnight incubation at 4 °C in rabbit polyclonal antibodies (Abcam Inc., Cambridge, UK) against FBN1 (ab231094, 1 : 1000), E‐cadherin (ab15148, 1 : 500), N‐cadherin (ab76057, 1 : 1000), Vimentin (ab137321, 1 : 2000), and GAPDH (ab9485, 1 : 2500). The 1‐h membrane incubation was then conducted in secondary antibody to goat anti‐rabbit IgG H&L (ab6721, 1 : 3000; Abcam Inc.) tagged by horseradish peroxidase at room temperature for 1 h. Then, the membrane was scanned and developed using a chemiluminescence instrument (Gel Company, San Francisco, CA, USA). The analysis of relative protein expression was conducted using image‐pro plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).