Anti ung

Cell pellets were incubated with RIPA buffer (25 mM Tris:HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Sigma-Aldrich), containing 1 × Halt Protease/Phosphatase Inhibitors (Thermo-Fisher Scientific) and incubated 10 min on ice. Lysates were centrifuged at 14,000g for 15 min at 4 °C and the supernatant stored at − 20 °C. Extracts were mixed with SDS-loading buffer, boiled, electrophoresed and transferred to PVDF membranes. Primary antibodies were incubated at 4 °C overnight.
The antibodies anti-DCTPP1 and anti-dUTPase are anti-rabbit polyclonal antibodies generated in our laboratory [10 (link), 11 (link)]. Antibodies directed against TK1 (C-4), dCK (H-3), DCTD (F-9), TMPK (B-8), TS (H-265) and R2 (A-5) were obtained from Santa Cruz; p53R2 (Sigma-Aldrich); SAMHD1 (ABIN3187981) from Antibodies Online; TK2 (Acris); pChk2-Thr68 (C13C1) and pChk1-Ser345 (133D3) from Cell Signaling technology; CTPS1 (Abcam); α-tubulin (B-5-1-2) and actin (MM2/193) from Sigma-Aldrich and anti-UNG (GeneTex).

Protein analysis by western blot was performed on human preadipocytes and Wt and miR‐424(322)/503‐null MEFs following adipogenesis. Cells were washed with cold PBS and lysed with protein lysis buffer (Cell Signaling Technology, 9803). Protein concentrations were determined using a protein assay kit (Life Technologies, 22660/22663) and equal amounts of proteins were subjected to SDS‐PAGE and transferred to nitrocellulose membranes (Whatman, 10600001). Non‐specific binding was blocked by incubation with TBST (20 mm Tris‐Hcl pH7.4, 150 mm NaCl, 0.1% Tween‐20) plus 5% of non‐fat milk (Bio‐Rad, 170–6404). Membranes were incubated overnight at 4 °C with a 3% BSA (Fisher BioReagents, BP9703‐100)‐TBST solution containing the primary antibody. Thereafter, membranes were incubated for 1 h with secondary HRP‐conjugated antibodies at room temperature (Jackson Immunoresearch, 115‐035‐003 and 111‐035‐003). Signal was detected with a western blot HRP substrate (Millipore, WBLUF0500). The antibodies used in this study include anti‐SNCG (Sigma Aldrich, HPA014404), Anti‐TNFAIP8 (Sigma Aldrich, HPA057089), anti‐UNG (GeneTex, GTX113860), anti‐CHRDL1 (GeneTex, GTX49225), anti‐CEBP‐α (Santacruz, sc‐166258) anti‐AdipoQ (Life Technologies, PA1‐24411), anti‐PPARγ (Santacruz, sc‐7273),anti‐FABP4 (R&D Systems, MAB3150) and anti‐Actin (Sigma Aldrich, A5441).