Ab108981

Antibodies used for Western blot, IHC, and IF are: Anti-SENP1 (ab108981; Abcam), JAK2 (3230 S; Cell Signaling Technology), SUMO2/3 (ab3742; Abcam), STAT3 (ab5073; Abcam), p-STAT3 (ab76315; Abcam), Bcl-xL (2764 S; Cell Signaling Technology), and β-actin (5441; Sigma-Aldrich).
Cisplatin (1134357; Sigma) was dissolved in sterile saline for cell survival and xenograft studies. Momordin Ic (A14773; Adooq Bioscience) was dissolved in DMSO.

Equal amounts of proteins were electrophoresed on SDS-PAGE gels and then transferred to a PVDF membrane. Membranes were probed overnight at 4°C with the following primary antibodies (all from Abcam): Sox2 (1:1000, ab97959), cTnI (1:2000, ab47003), SENP1 (1:1000, ab108981), SENP2 (1:1000, ab131637), SENP3 (1:1000, ab71677), SENP5 (1:1000, ab58420), SENP6 (1:1000, ab77619), SENP7 (1:200, ab224752), GAPDH (1:1000, ab8245), Bcl-2 (1:1000, ab692), and caspase-3 (1:1000, ab2302). Primary antibody binding was detected by secondary antibodies and visualized by the enhanced chemiluminescence method.

To investigate the correlation of expression of HIF’s and SENP1 genes, the publicly available Human Protein Atlas database of RCC samples (N = 877) were analyzed for the correlation (R^2) or covariation by Spearman correlation methods for RNA expression of HIF1α, HIF2α, and SENP1 genes. For tissue microarray (TMA) analysis, malignant and tumor-adjacent benign tissues were used to construct a manual tissue array [43 (link)]. A total of 471 malignant cores and 190 benign cores were included. Immunohistochemistry was performed by UW Translational Research Initiatives in Pathology (TRIP) facility. TMA with human samples was performed the protocol (#2011-0179) approved by IRB. Anti-SENP1 (ab108981), anti-HIF1α (ab51608), or anti-HIF2α (ab109616), all from Abcam, MA, was applied to the TMAs, and hematoxylin was used for count-staining. Stained slides were scanned by Vectra slide scanner (PerkinElmer, MA) and SENP1, HIF1α, and HIF2α expression levels in the nuclears (where SENP1 would act on these HIF proteins) were quantified and analyzed in inForm software (PerkinElmer).

Complete and clear paraffin-embedded heart sections were selected. Sections were dewaxed with xylene and treated with an alcohol gradient. Next, the paraffin-embedded sections were placed in 0.01 M citrate buffer and autoclaved for antigen retrieval. An endogenous peroxidase blocker (reagent A) was added dropwise, followed by incubation at room temperature for 10 min in the dark to inactivate endogenous peroxidase activity. Goat serum was added dropwise and allowed to stand at room temperature for 30 min. Then, the primary antibody was added dropwise, followed by overnight incubation at 4°C. Primary antibodies (all from Abcam) against SENP1 (1:200; ab108981), SENP2 (1:200; ab58418), SENP3 (1:100; ab71677), SENP5 (1:200; ab58420), and SENP7 (1:200; ab224752) were used. The sections were then incubated with horseradish peroxidase-labeled polymer (reagent B) for 30 min at 37°C. DAB color development was applied for 3–5 min, and the sections were observed under a microscope until the section showed a caramel color. Hematoxylin staining of the nucleus was conducted for 4 min, followed by treatment with 1% hydrochloric acid alcohol, saturated lithium carbonate, alcohol, and xylene in that order. Finally, a suitable amount of neutral resin was applied on the sections that were then covered with a coverslip. Photographs were obtained under the OLYMPUS microscope.

Fifty micrograms of protein obtained from each sample (BRPCs) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad miniature slab gel apparatus and electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked in a 5% nonfat dried-milk solution and incubated overnight with rabbit anti-SENP1 (ab108981, 1 : 1000, Abcam, USA), rabbit anti-PARP (#9542, 1 : 1000, Cell Signaling Technology, USA), rabbit anti-BCL-2 (#2870, 1 : 1000, Cell Signaling Technology, USA), mouse anti-HA (ab18181, 1 : 1000, Abcam, USA), or Flag antibody (F1804, 1 : 1000, Sigma, USA). Blots were stained with mouse anti-β-actin (A5441, 1 : 1000, Sigma, USA) (as an internal control) to confirm equivalent total-protein loading.

Antibodies to SENP1 (C-12), Lck (3A5), RanGAP1 (C-5), c-Myc (9E10), β-actin (C4), and GAPDH (FL-335) were from Santa Cruz Biotechnology. Antibodies to SENP1 (ab108981), SUMO1 (ab32058), and SUMO2/3 (ab3742) were from Abcam. Antibodies to HA (C29F4) and glutathione S-transferase (GST) were from Cell Signaling Technology. M2 antibody to Flag (F1804) was from Sigma-Aldrich, and 4G10 (05-1050) was from Millipore. Anti-human CD3 (OKT3) and anti-human CD28 (CD28.2) were from eBioscience. Goat anti-mouse IgG (31160) was from Thermo Fisher Scientific. Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch.