Ab56053

Immunoblotting was performed as previously reported^{7 (link),10 (link)}. The following antibodies were used for detection: rabbit anti-NAMPT (#A300-372A, Bethyl Laboratories, Inc., 1:20,000), rabbit anti-PARP-1 (#ALX-210-302-R100, Enzo Life Sciences, 1:4000), rabbit anti-CDA (#ab56053, Abcam, dilution 1:500), rabbit anti-BLM (#ab2179, Abcam, dilution 1:5000), rabbit anti-β-actin (#A2066, Sigma-Aldrich, 1:5000), rabbit anti-HSP90 (#ab2928, Abcam, 1:5000), rabbit anti-His-tag (#66,005-I, Proteintech, dilution 1:1000), mouse anti-GAPDH (#G8795 1:1000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#A9169, Sigma-Aldrich, 1:5000), and HRP-conjugated goat anti-mouse IgG (#A3682, Sigma-Aldrich, 1:5000). Bands were visualized by chemiluminescence (Clarity Western ECL Substrate, Bio-Rad), with a ChemiDoc XRS+ Molecular Imager and Image Lab Software (Bio-Rad).

Cells were lysed in 8 M urea, 50 mM Tris HCl, pH 7.5 and 150 mM β-mercaptoethanol, sonicated and heated at 75°C for 10 minutes. Samples (equivalent of 2 x 10⁵ cells) were subjected to electrophoresis in NuPAGE Novex 4–12% Bis-Tris pre-cast gels (Life Technologies). The procedures used for gel electrophoresis and immunoblotting have been described elsewhere [16 (link)]. Primary and secondary antibodies were used at the following concentrations: rabbit anti-BLM antibody (1:5,000; ab2179 from Abcam); rabbit anti-CDA antibody (1:500; ab56053 from Abcam); rabbit anti-β-actin antibody (1:10,000; Sigma); rabbit anti-PARP-1 antibody (1:4,000; ALX-210-302 from Enzo Life Sciences); rabbit anti-Chk2 (1/500; 2662 from Cell Signaling; rabbit anti-Chk2 T68 (1/500; 2661 from Cell Signaling; rabbit anti-H2AX (1/500; 2595 from Cell Signaling); rabbit anti-H2AX S139 (1/500; 2577 from Cell Signaling); horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; Santa Cruz Biotechnology).

Proteins were isolated in lysis buffer (8 M urea, 50 mM Tris-HCl, pH 7.5, and 150 mM β-mercaptoethanol), separated by electrophoresis in 4–12% Bis-Tris pre-cast gels (NuPAGE Novex, Life Technologies), and blotted onto polyvinylidene fluoride membranes, which were then incubated with appropriate primary and secondary antibodies. Bands were visualized by chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) with a ChemiDoc XRS+Molecular Imager and Image Lab software. Uncropped immunoblots are presented in Supplementary Fig. 5. The following antibodies were used for detection: mouse anti-Tau-5 (#ab80579, Abcam, dilution 1:1000), mouse anti-Tau-1 (clone PC1C6, #MAB3420, Millipore, dilution 1:1000), rabbit anti-CDA (#ab56053, Abcam, dilution 1:500), rabbit anti-BLM (#ab2179, Abcam, dilution 1:5000), rabbit anti-UBTF (#sc-9131, H-300, Santa Cruz, dilution 1:500), rabbit anti-γH2AX (S139) (#2577, Cell Signaling, dilution 1:500), mouse anti-GAPDH (#G8795, Sigma-Aldrich, dilution 1:10,000), rabbit anti-β-actin (#A2066, Sigma-Aldrich, dilution 1:10,000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#sc-2054, Santa Cruz, dilution 1:5000), and HRP-conjugated goat anti-mouse IgG (#sc-2055, Santa Cruz, dilution 1:5000).