Ab8224

CellTiter Glo (Promega) viability assays and clonogenic assays were performed as in reference [13 (link)], using cultures growing in full medium supplemented with 10% FCS. Western blotting was performed as described [13 (link)], using antibodies to: IGF-1R (#3027, Cell Signaling Technology, CST), phospho-Y1135/6 IGF-1R (#3024, CST), phospho-S473 AKT (#4051, CST), total AKT (#9272, CST), phospho-T202/Y204 ERK 1/2 (#4377, CST), total ERK 1/2 (#4695, CST), MGMT (#557045, BD Pharmingen), p53 (#9282, CST) PARP (#9542, CST) and actin (ab8224 or ab8227, Abcam). For each western blot shown, similar results were obtained in 1–2 further independent replicates. Viability and survival data were graphed and curve-fitted (GraphPad Prism v5) to interpolate GI₅₀ and SF₅₀ values (drug concentrations that suppress growth or survival to 50% of control values). Apoptosis was quantified by Apo-ONE^® Homogenous Caspase 3/7 Assay (Promega).

Two replicate western blot experiments were performed to test for apoptosis markers in X. laevis with 3q29 gene knockdown (S12 Fig). Embryos at stages 20–22 were lysed in buffer (50mM Tris pH 7.5, 1% NP40, 150mM NaCl, 1mM PMSF, 0.5 mM EDTA) supplemented with cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Basel, Switzerland). Blotting was carried out using rabbit polyclonal antibody to cleaved caspase-3 (1:500, 9661S, Cell Signaling Technology, Danvers, MA, USA), with mouse anti-beta actin (1:2500, AB8224, Abcam, Cambridge, UK) as a loading control on a Mini-PROTEAN TGX precast 4–15% gradient gel (Bio-Rad, Hercules, CA, USA). Chemiluminescence detection was performed using Amersham ECL western blot reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Band intensities were quantified by densitometry in ImageJ and normalized to the control mean relative to beta-actin. Due to the low number of replicates, we did not perform any statistical tests on data derived from these experiments.

After cells were lysed, electrophoresis of equal amounts of protein was conducted on a 12% SDS-polyacrylamide gel. Separated proteins were transferred to polyvinylidene fluoride membranes, which were then blocked for 1 h at room temperature using 5% skim milk in phosphate-buffered saline containing 0.1% Tween-20 (PBST). Then, the membranes were incubated overnight at 4°C with the following primary anti-bodies: TPM1 (1:1000, 3910; Cell Signaling Technology), α-SMA (1:250, ab7817; Abcam, Cambridge, United Kingdom), Col1a1 (1:1000, ab6308; Abcam), β-catenin (1:1000, 8480; Cell Signaling Technology), GSK3β (1:1000, 9315; Cell Signaling Technology), p-GSK3β (Ser9) (1:1000, ab131097; Abcam), and β-actin (1:1000, ab8224; Abcam). After the membranes were washed three times with PBST, they were incubated for 2 h at room temperature in either HRP-goat-anti-rabbit (ab6721, Abcam) or HRP-goat-anti-mouse (ab6789, Abcam) secondary antibodies. After membranes were washed with PBST three times, immunoreactive bands were visualized using Pierce ECL Plus western blotting substrate (32132; Thermo Fisher Scientific, Waltham, MA, United States) and quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, United States). Lamin B1 and β-actin were used as internal loading controls.

Adult flies (day 5) were lysed as previously described (Wang et al., 2011 (link); Tsai et al., 2014 (link)). Lysates were analyzed by SDS-PAGE and immunoblotted with mouse anti-ATP5β (ab14730; AbCam) at 1:3000, mouse anti-β-actin at 1:3000 (ab8224; AbCam), guinea pig anti-DMiro (GP5) at 1:20,000 (Tsai et al., 2014 (link)), mouse anti-milton at 1:100 (Stowers et al., 2002 (link)), rabbit anti-VCP (SAB1100655; Sigma–Aldrich) at 1:5000, or rabbit anti-Marf (Ziviani et al., 2010 (link)) at 1:2000, and HRP-conjugated-goat anti-guinea pig, rabbit, or mouse IgG (Jackson ImmunoResearch Laboratories) at 1:5000.

Strains IF235 (sir2Δ), IF140 and IF230 (sir2 over-expression; sir2OE) were grown in synthetic complete media (Sunrise Scientific) to OD 1.0, and whole cell extracts were prepared by trichloroacetic acid precipitation with bead lysis (Keogh et al., 2006 (link)). These cell extracts were combined with loading buffer, boiled for 5 min at 95°C, and resolved via a 4–20% TGX SDS-PAGE gel (Bio-Rad). Proteins were transferred to a PVDF membrane, and then blocked with PBS Odyssey blocking buffer (Li-Cor, Inc.) for one hour with shaking at 22°C. The membrane was incubated overnight at 4°C while shaking in phosphate buffered saline +0.05% Tween-20 (PBST) containing 1:1000 dilution of anti-β actin antibody (mouse, ab8224, Abcam) and 1:500 dilution of an S. pombe-specific anti-Sir2p antibody (rabbit, generously provided by Dr. Allshire) (Buscaino et al., 2013 (link)). The membrane was washed once in PBST, and then agitated for 4 hr at 22°C with 1:10,000 dilution of goat anti-mouse IgG and 1:10,000 dilution of goat anti-rabbit IgG (IRDye 680RD and IRDye 800CW, respectively, Li-Cor, Inc.). The membrane was washed three times with PBST and imaged on an Odyssey CLx dual-channel imaging system (Li-Cor, Inc.).

Yeast protein extracts were prepared as previously described [52 (link)]. In brief, cells in early-log phase were harvested at 5000×g for 10 min at 4°C. The cell pellet was washed twice in cold water and resuspended in 0.1 M NaOH. Following 5 min alkaline treatment at room temperature, the cells were centrifuged at 10000×g for 1 min and the supernatant was discarded. The cell pellets were resuspended in Laemmli SDS sample buffer (Alfa Aesar), incubated for 5 min at 95°C and centrifuged at 10000×g for 30 s to remove cell debris; 0.50 mg of wet cell weight was analyzed by immunoblotting. Primary antibodies were rabbit anti-HA-tag (Abcam, ab9110, 1:3000), rabbit anti-NAA30 (Sigma–Aldrich, HPA057824, 1:1000), rabbit anti-NAA60 (Sigma–Aldrich, SAB1102546, 1:1000) and mouse anti-actin (Abcam, ab8224, 1:5000). Secondary antibodies were HRP-conjugated anti-rabbit and anti-mouse (both from GE Healthcare, NA934 and NA931, 1:5000).