3xflag peptide

Cell lysates were prepared in Pierce IP lysis buffer (Thermo; 87788). Total protein concentration was determined by BCA assay and 500 μg lysate was incubated with anti-MRE11 (ab214; abcam, Cambridge, UK) at 4 °C overnight on a tube rotator and then added to protein A/G plus agarose (sc2003; Santa Cruz, Dallas, TX) for 1 h at 4 °C. The beads were washed five times in PBS-Tween 0.05% and binding proteins eluted by boiling (95 °C for 10 min) in 40 μL Laemmli buffer before SDS-PAGE gel running. Ten percent of the total lysate was retained as the load fraction. The antibodies used were anti-MRE11 (ab30725; abcam, Cambridge, UK), anti-RAD50 (3427; CST, Danvers, MA), anti-NBS1 (NB100-143; Novus Biological, Centennial, CO), anti-myc-tag (2278, CST, Danvers, MA). To isolate SPRTN-interacting proteins, whole cell lysates were prepared in lysis buffer (50 mM Tris-HCl pH7.4; 150 mM NaCl; 0.1% NP-40; 1 mM EDTA, and 5% glycerol; protease and phosphatase inhibitors) containing 500 U/ml of benzonase at 4 °C. Flag-tag protein complexes were separated using the anti-flag M2 antibody (F1804; Merck, Whitehouse Station, NJ) and washed five times in IP buffer containing 0.05% NP-40 and eluted in 3xFlag peptide (F4799; Merck) for 30 min at RT.

PrimPol was isolated from HEK293 cells as described previously (29 (link)). Briefly, approximately 1 × 10⁷ cells expressing Flag-tagged PrimPol were lysed in NETN buffer [150 mM NaCl, 30 mM tris (pH 7.5), 0.5% NP-40, 2.5 mM MgCl₂, and deoxyribonuclease I (100 μg/ml)] for 30 min at 4°C. Cell lysate was incubated with Anti-Flag M2 magnetic beads (Merck) for 2 hours at 4°C before beads were washed three times with wash buffer [150 mM NaCl, 30 mM tris (pH 7.5), and 0.1% NP-40]. Proteins and interacting partners were eluted with 50 μl of elution buffer [25 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 3xFLAG peptide (200 μg/ml; Merck)] and analyzed against input cell lysate by Western blot.
For mass spectrometry analysis, 10 × 10⁷ cells expressing Flag-tagged PrimPol were lysed in RIPA buffer. Protein was bound by Anti-Flag M2 magnetic beads (Merck) for 2 hours at 4°C before being washed three times in 50 mM ammonium bicarbonate. After overnight on-bead digestion at 37°C by Glu-C protease, the peptides were analyzed on an Orbitrap Exploris 480 [with FAIMS (Field asymmetric Ion mobility spectrometry)] mass spectrometer by the Proteomics Core Facility at CEITEC (Brno, Czech Republic).

HEK293 cells were transfected with PGK::FLAG-GIG or PGK::FLAG-BTB constructs. 28 hours later, cells were lysed and immunoprecipitation was performed as previously described [29 (link)]. Targets were eluted by incubation with 3X flag peptide (Sigma). Samples were analyzed by Applied Biomics, Inc (Hayward, CA) via NanoLC-MS/MS after tryptic digestion, and only high confidence hits (CI > 95%) were considered for analysis. CRAPome analysis was performed as previously described [30 (link)]. DAVID analysis was performed using the published online tool [17 (link)]. For follow-up immunoprecipitation experiments, the same strategy was used to isolate flag-GIG. Endogenous GIG was immunoprecipitated using rabbit α GIG (NBP1-49924, Novus Biologicals). Endogenous VIM was immunoprecipitated using mouse α VIM (BD 550513 or AMF-17b). The mouse peripherin construct was a generous gift from Jean-Pierre Julienne (Université Laval).