Multi gauge

Immunoblotting and IP experiments were performed as described before19 (link). Nocodazole was used at 100 ng/ml for 16 hours. The following primary antibodies were used for immunoblotting: anti-Ataxin-10, anti-Aurora B, anti-β-actin, anti-HA and anti-FLAG M2 (Sigma). Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000, and analyzed using Multi Gauge (Fujifilm).

After the careful removal of peri-aortic soft tissue, the whole aorta was saline-perfused and excised. The aorta was homogenated, and protein lysates were subjected to SDS-PAGE followed by transfer onto a PVDF membrane. Membranes were probed with monoclonal antibodies against p-JNK (CST, #9251), JNK (CST, #9252), p-ERK (CST, #9106), ERK (CST, #4695), p-P65 (CST, #3033), VEGF (BD Biosciences, 555036), intercellular adhesion molecule (ICAM, Santa Cruz, SC-1511), vascular cell adhesion molecule (VCAM, Santa Crus, SC-1504), HIF-1α (GeneTex, GTX127309), total eNOS (CST, #9586) and phosphorylated eNOS (p-eNOS, #9574), Kruppel-like factor (KLF4, CST, #4038), SIRT1-mouse specific (CTS, #3931), SIRT1-human specific (CST, #2496), MMP-2 (CST, #4022), MMP-9 (CST, #G657) and β-actin. Bands were visualized by chemiluminescence detection reagents. Densitometric analysis was conducted with imaging processing software (Multi Gauge, Fujifilm), and data were expressed as a fold change relative to the controls.

Small tumors, pieces of tumors, or pooled islets (2 mice per lane) were disrupted in RIPA buffer (150 nM NaCl, 1% Triton-X, 50 mM Tris, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with Complete, ethylenediaminetetraacetic acid (EDTA)-free Protease inhibitors (Roche) and Halt Phosphatase inhibitors (Pierce) using the TissueLyser (Qiagen), followed by sonication. Cells were washed in PBS, then collected in RIPA buffer and sonicated directly. Protein concentrations were measured by bicinchoninic acid assay. Laemmli buffer was added to samples, equal protein amounts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred by electroblotting and membranes blocked in 5% milk/Tris buffered saline with Tween 20 (TBS-T) for 1 h. Membranes were incubated with appropriate antibodies overnight at 4 °C, then exposed to secondary antibodies for 1 h at room temperature and developed using ECL Western Blotting Substrate. Band density was evaluated using MultiGauge (Fujifilm) and normalized to beta actin or tubulin levels. For blots intended to be compared to each other, data were additionally normalized to a calibrator sample that was equally loaded on each blot. Uncropped western blots can be found in Supplementary Figures 7–10.