Succinyl lysine

PCa cell lines were lysed using radioimmunoprecipitation assay buffer (Catalog No. 89900, Thermo Fisher Scientific, Waltham, MA). Protein concentrations were measured by BCA assay (Catalog No. 23224, Thermo Fisher Scientific). Samples were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) for 90 min and transferred to polyvinylidene fluoride blotting membranes (0.2 μm pore size; GE Healthcare, Chalfont St. Giles, UK) for 2 h. The membrane was blocked in 5% bovine serum albumin for 2 h. Primary antibodies against SIRT1 (Catalog No. 8469S, Cell Signaling Technology Technology, Beverly, MA), SIRT2 (Catalog No. 12672S, Cell Signaling Technology), SIRT3 (Catalog No. 5490S, Cell Signaling Technology), SIRT4 (Catalog No. ab231137, Abcam, Cambridge, UK), SIRT5 (Catalog No. 8782S, Cell Signaling Technology), SIRT6 (Catalog No. 12486S, Cell Signaling Technology), SIRT7 (Catalog No. 5360S, Cell Signaling Technology) and succinyl-lysine (PTM Biolabs, Chicago, IL) were used to capture SIRT1-SIRT7 proteins overnight at 4 °C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, the membranes were washed in Tris-buffered saline-0.1% Tween 20 (TBS-T) three times for 15 min. ECL prime Western blotting detection reagent (RPN2232, Citiva) was used to visualize the blots.

Cells were harvested and lysed in TRIS‐HCL pH 7.4 with 1% triton X‐100 containing protease inhibitors and deacylase inhibitors (1 μM trichostatin A and 20 mM nicotinamide). Lysates were sonicated 5 times 2 seconds at 40 kHz amplitude on ice. Protein concentrations were determined using Pierce BCA protein assay kit (Thermofisher) and equal protein amounts were loaded on NuPAGE 4% to 12% gels (Invitrogen), transferred to nitrocellulose membrane, blocked in 3% BSA in PBS with 0.1% Tween‐20 at room temperature and incubated overnight with antibodies in the same buffer at 4°C. Primary antibodies used: β‐actin (#A5441, Sigma‐Aldrich), propionyllysine (#201, PTM biolabs), succinyllysine (#401, PTM biolabs), acetyllysine (#9441, Cell Signalling), histone 3 propionyllysine 23 (#613987, Active Motif), histone 3 acetyllysine 23 (#07‐355, Millipore). IR‐dye based secondary antibodies (LICOR) were used to detect antibody signals using Odyssey scanner (LICOR).

The primary antibodies used for immunohistochemistry, immunofluorescence, immunoprecipitation, and western blotting analysis were as follows: CDCP1 (Cell Signaling Technology #4115; Danvers, MA, USA), CDCP1 (Abcam #188818; Cambridge, UK), IdU (Abcam #181664), BrdU (Abcam #6326), HA (Abcam #18181), myc (Abcam #32072), Kras (Santa Cruz Biotechnology #sc-30; Dallas, TX, USA), Flag (Sigma-Aldrich #F1084), malonyl-lysine (PTM Biolabs 901; Chicago, IL, USA), succinyl-lysine (PTM Biolabs 401), glutaryl-lysine (PTM Biolabs 1151), acetyl-lysine (PTM Biolab 104), Sirt5 (Cell Signaling Technology 8782), tubulin (Abcam 7291), and TPI (Abcam #96696). The secondary antibodies used for immunofluorescence were Alexa Fluor 488 or 594 goat anti-mouse (#A-11001, #A-11005) or rabbit anti-mouse (#A-11034, R37117) IgG from Thermo Fisher Scientific (Waltham, MA, USA). The HRP-conjugated secondary antibodies (#7076, #7074, and #7077) used for western blot were from Cell Signaling Technologies.