Ab91459

The antibodies employed for this work were as follows. Primary: Rat anti‐CldU (BioRad, OBT0030G; 1:200), mouse anti‐H2AX‐phosphoSer139 (Millipore, clone JBW301; 1:500) rat anti‐BrdU (Abcam, ab6326; 1:30), mouse anti‐BrdU (Becton Dickinson, 347580; 1:25), mouse anti‐single‐strand DNA (Millipore, MAB3034; 1:25), mouse anti‐UNG (Origene, TA503755; 1:500), rabbit anti‐vinculin (Abcam, ab91459; 1:5,000), rat anti‐tubulin (Abcam, ab6160; 1:5,000). Secondary: Goat anti‐rat Alexa Fluor 488 (Thermo Fisher, A11006; 1:1,000), donkey anti‐mouse Alexa Fluor 568 (Thermo Fisher, A10037; 1:1,000), goat anti‐mouse Alexa Fluor 488 (Thermo Fisher, A32723; 1:25), donkey anti‐rabbit Alexa Fluor 488 (Thermo Fisher, A21206; 1:1,000), goat anti‐rat Alexa Fluor 568 (Thermo Fisher, A11077; 1:25), donkey anti‐mouse Alexa Fluor 647 (Thermo Fisher, A31571; 1:25).

Co-immunoprecipitation (Co-IP) was performed by a specific, commercially available polyclonal anti-Vinculin antibody (ab91459, Abcam). After overnight incubation with antibody at 4 °C, 5 (T-Nu, SC-Nu) or 20 μL (T-Cy, SC-Cy) of Dynabeads with conjugated Protein A (Dynabeads Protein A Immunoprecipitation Kit, Novex) was added, and the procedure was carried out according to the manufacturer’s recommendations. In the control experiment, the rabbit IgG polyclonal isotype control (ab37415, Abcam) was applied to determine the non-specific proteins bound to the beads. Co-IP samples were resuspended in 100 mM TEAB containing 2% SDC. Cysteines were reduced with a 10 mM final concentration of TCEP and blocked with a 40 mM final concentration of chloroacetamide (60 °C for 30 min). Samples were cleaved on beads with 1 µg of trypsin at 37 °C overnight. After digestion, the samples were centrifuged, and the supernatants were collected and acidified with TFA to a final concentration of 1%. SDC was removed by extraction to ethyl acetate [53 (link)]. Peptides were desalted using in-house-produced stage tips packed with C18 disks (Empore) according to Rappsilber et al. [54 (link)].

RIPA buffer with proteinase inhibitors was employed for cell lysis. Bradford reagent (Sigma; Merck) was adopted to measure protein concentrations. A total 20-μg protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then the proteins were transferred onto nitrocellulose membranes, followed by 1-h blocking with 5% non-fat milk at room temperature. Antibodies against KLF13 (18352-1-AP, proteintech), IFIT1 (23247-1-AP, proteintech), and Vinculin (ab91459, abcam) were incubated with the membranes overnight at 4 °C, followed by incubation with secondary antibodies for 2-h at room temperature.