Ab32058

Cells were dissolved in IP buffer for 30 min, and then the cell lysate was ultrasonically centrifuged at 20000 × g and 4°C for 10 min. The supernatant was incubated with antibody and protein A/G beads at 4°C and eluted in 1% SDS solution. The antibodies used in Western blot analysis included anti-rabbit PIAS1 (2 μg, 23395-1-AP, Thermo Fisher Scientific), NFATc1 (1: 50, #8032S, CST), GAPDH (1: 60, ab181602, Abcam, Cambridge, UK), IgG (ab210935, Abcam), anti-Flag (F3165, Sigma-Aldrich), anti-HA (MMS-101P, Covance, Princeton, NJ), and anti-SUMO1 (ab32058, Abcam).

Protein from microglia or ischemic penumbra was centrifuged at 13,000 × g at 4°C for 30 min and quantified through a BCA kit (P0011, Beyotime Biotech). Equal amounts of protein were loaded onto 10% SDS‐PAGE gel (PG212, Epizyme), separated by electrophoresis and transferred to Polyvinylidene fluoride membranes (PVDF, 1620177, Bio‐Rad). The membrane was blocked by 5% non‐fat milk for 2 h and incubated with primary antibodies against IL‐1β (sc52012, Santa Cruz), IL‐6 (sc‐28343, Santa Cruz), TNF‐α (sc‐52746, Santa Cruz), iNOS (BS40374, Bioworld Biotechnology), p‐NF‐κB/p‐P65 (3033, Cell Signaling Technology), NF‐κB/P65 (8242, Cell Signaling Technology), IKKα/β (sc‐8032, Santa Cruz), p‐IKKα/β (2697, Cell Signaling Technology), IκBα (BS3601, Bioworld Biotechnology), p‐IκBα (2859, Cell Signaling Technology), NEMO (ab178872, abcam), anti‐Ubiquitin (ab134953, abcam), anti‐SUMO (ab32058, abcam), COX‐2 (BS1076, Bioworld Biotechnology) or β‐actin (BS40736, Bioworld Biotechnology) overnight at 4°C. On the following day, the membrane was immersed in the corresponding secondary antibodies (BS22357/BS22356, Bioworld Biotechnology) on the shaker for 1–2 h. After moistened with the enhanced chemiluminescence (ECL, 34580, Thermo Fisher Scientific), the protein band was visualized through Gel‐Pro system (Tanon Technologies) and analyzed using ImageJ (ImageJ 1.5, NIH).

Standard immunohistochemical (IHC) staining and the quantitation of the staining were performed as previously described [15 (link)]. Briefly, after de-waxing of the 5μm thick sections using xylene and re-hydration with ethanol, endogenous peroxidase activity was blocked using 3% hydrogen peroxide. This was followed by antigen retrieval, blocking with 10% normal serum, and incubation of the sections with anti-SAE1 (1:500; #ab185552, Abcam, Cambridge, UK), anti-SUMO1 (1:100; #ab32058, Abcam), anti-SUMO2 (1:100; #ab212838, Abcam), and UBC9 (1:100; #ab75854, Abcam) antibodies overnight at 4 °C, followed by goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibody (1:10,000; #65-6120, Thermo Fisher Scientific Inc., Waltham, MA, USA). As chromogenic substrate, Diaminobenzidine (DAB) was used, and the stained sections were counter-stained with Gill’s hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA). The univariate and multivariate analyses were done using the Cox proportional hazards regression model.

The fresh anterior lens capsules were immediately fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 20 min at room temperature, and treated with 3% hydrogen peroxide/deionized water buffer to inhibit endogenous peroxidase. Then, the fixed capsules were blocked with 10% normal goat serum in PBS and then incubated with anti-SUMO-1 (ab32058; Abcam, USA) antibody in PBS supplemented with 10% normal goat serum overnight at 4°C. Secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology, USA) was then applied for 1 h at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB; Cell Signaling Technology, USA) and then counterstained with hematoxylin and coverslipped with permount. Immunostaining images were captured using a fluorescence microscope (TH4-200; Olympus, Japan).

Proteins were separated on a 10% or 12% SDS-PAGE gel to test the target protein level using specific antibodies. The specific primary antibodies included SAE1 (ab56957 & ab185552, Abcam), SUMO1 (ab32058, Abcam), Akt (4961, Cell signaling), p-Akt (4060, Cell signaling), CDK2 (ET1602–6, HuaBio), Cyclin D1 (2922, Cell signaling), p21 (ab109199, Abcam), Bcl-2 (ET1603–11, HuaBio) and active Caspase-3 (ET1602–47, HuaBio), which were used to detect SAE1-invoved cell signaling pathway. The corresponding secondary antibody was subsequently incubated for 1 h to visualize signals at room temperature. The mouse anti-β-actin antibody (TA-09, ZSGB) was taken for signal normalization.