Prestige antibody

Polyclonal rabbit anti-GWL antibodies were generated and purified by Eurogentec. Mouse anti-Flag M2, rabbit [Prestige Antibodies] and mouse (RIPLY 74C) anti-MASTL (GWL) antibodies were purchased from Sigma-Aldrich and Abcam respectively. Polyclonal rabbit anti-phospho(Ser67)-ENSA/ARPP19 antibody was generated by Generon. Secondary antibodies were HRP-conjugated, polyclonal goat-derived antibodies against mouse and rabbit [Dako, Agilent Technologies].

Protein extracts were loaded into 4–20% or 10% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, 4561096). Then, it was transferred to polyvinylidene difluoride PVDF membrane for 30 min at 15 V using a semi-dry electrophoretic transfer cell (Bio-Rad, Watford, UK). Blots were probed with the recommended concentration of primary Ab (1:500–1:1000) except for FLYWCH1 (1:200) was used (Prestige Antibodies, Sigma-Aldrich, Gillingham, UK, HPA041001). As suitable, antibodies against β-actin or β-tubulin were used as a loading control.

Progression tissue microarray (TMA) slides were provided by the NCI CDP and included 98 benign nevi, 73 primary tumors, and 41 lymph node metastases and 72 metastases from other locations. Other investigators may have received slides from these same array blocks. Immunohistochemical staining was performed on the TMA samples using a commercially available polyclonal rabbit anti-ICAM-1 antibody (Prestige Antibodies, Sigma Aldrich, Rehovot, Israel) according to standard procedures. A blinded assessment of ICAM-1 expression was conducted by an expert pathologist (IB). For each sample, intensity of ICAM-1 membrane expression was scored as none, low intensity, intermediate or high. Digital images were captured with Olympus BX51 microscope.

We prospectively collected clinical and pathological data from consecutive consenting patients undergoing radical prostatectomy for prostate cancer between 2014 and 2015. Surgical specimens underwent conventional histological processing and examination according to the Stanford protocol. Additionally, in each tissue section, areas of adenocarcinoma were identified and graded according to Gleason Score. Then, the Index lesion corresponding to the highest Gleason Score was studied by immunohistochemistry, using anti-SIRT7 antibody (Prestige Antibodies^®, SIGMA-ALDRICH^®). SIRT7 expression was analyzed in a qualitative manner by evaluating positivity or negativity of expression, comparing healthy and tumor areas, and in a quantitative manner. To quantify the expression of SIRT7 we used the Allred Score (0 to 8) [10 (link)] obtained by adding proportion of staining tumor (0 to 5) and signal intensity (0 to 3). The quantification was done by determining the average intensity and proportion of nuclei labelled including the nucleolus.
In each index lesion, the Allred Score and Gleason Score were compared using the Student t test (significance with p < 0.05). The analysis was performed blindly by two independent investigators.

Whole cell extracts were separated by 8% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane and probed with anti–AURKA (C4718T, 1:1000, Cell Signaling Technology); anti-CCNA2 (H432, 1:1000, Santa Cruz Biotechnology); anti-CCNB1 (ab72, 1:1500, Abcam); anti-IDH1 (8137S, 1:1000, Cell Signaling Technology); anti-PHGDH (13428S,1:1000, Cell Signaling Technology); anti-PKM (3190S, 1:1000, Cell Signaling Technology); anti-TOP2B (HPA024120, 1:250, Prestige antibodies, Sigma); anti-TUB (T6074,1:400000, Sigma) at 4°C, overnight. 12.5% gels were used similarly and probed with anti-Total H3 (ab1791, 1:5000, Abcam); anti-phosphoH3 (ab5176, 1:1000, Abcam). Secondary antibodies, donkey-anti-mouse (715-035-151, 1:10000, Jackson ImmunoResearch Laboratories) or anti-rabbit (711-035-152, 1:10000, Jackson ImmunoResearch Laboratories) were conjugated to horseradish peroxidase antibodies and incubated for 1 h at room temperature. Blots were visualized with the Pierce ECL Western blotting substrate (34087, Thermo Scientific) according to manufacturer’s instructions.