Sc 373750

Antibodies used were as follows: anti-FANCD2, 1:100 dilution (sc-20022, Santa Cruz Biotechnology); anti-FANCI, 1:500 dilution (FARF); anti-Flag, 1:1,000 dilution (M5, F4042, Sigma-Aldrich); anti-UHRF1, 1:1,000 dilution (sc-373750, Santa Cruz Biotechnology); anti-α-Tubulin, 1:2,000 dilution (05-829, Merck-Millipore), anti-ATR, 1:100 dilution (ab2905, abcam); anti-pChk (S345), 1:1,000 dilution (133D3, Cell Signaling Technology), anti-USP1, 1:1,000 dilution (14346-1-AP, Proteintech).

HeLa (originally from ATCC) and PD20 (kindly provided by the FARF repository) cells were grown in DMEM (D5796, Sigma) supplemented with 2.5–10% fetal bovine serum. Antibodies used were as follows: anti-FANCD2, 1:100 dilution (sc-20022, Santa Cruz Biotechnology); anti-FANCI, 1:1,000 dilution (FARF); anti-FANCA, 1:1,000 dilution (FARF); anti-Lamin B, 1:1,000 dilution (sc-6216, Santa Cruz Biotechnology); anti-Flag, 1:1,000 dilution (M5, F4042, Sigma-Aldrich); anti-Ubiquitin, 1:400 dilution (FK2,BML-PW8810, Enzo Life Sciences); anti-UHRF1, 1:1,000 dilution (sc-373750, Santa Cruz Biotechnology); and anti-a-Tubulin, 1:2,000 dilution (5829, Millipore).

Total lysates were prepared from cells using NP40 lysis buffer (50 mM Tris-HCL [pH 8.0], 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% NP40, 0.5 × protease inhibitor cocktail, 1 mM EDTA [pH 8.0]). The lysates were rotated 30 min at 4 ℃, sonicated on ice for 10 s and centrifuged. The lysates were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membranes. The membranes were probed at 4 ℃ overnight with the primary antibodies anti-UHRF1 (1:1000, sc-373750, Santa Cruz), anti-TIP60 (1:500, sc-166323, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-mouse IgG (1:1000, sc-2025, Santa Cruz), anti-FLAG® M2 (1:10000, F3165, Sigma), anti-Rabbit IgG (1:5000, 12–370, Merck) and anti-β-actin (1:1000, sc-47778, Santa Cruz). The blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG (1:5000, Enzo Life Sciences) and detected using an ECL system (ABClon).

Mice were injected i.p. with 50 mg/kg of EdU in PBS and lungs were harvested 16 h after injection. EdU and UHRF1 protein expression was evaluated using the Click-IT Edu Alexa Fluor 488 Imaging kit (C10337, Invitrogen) in formalin-fixed and paraffin-embedded lung tissue sections. After paraffin removal, endogenous peroxidase activity was inhibited with 3% hydrogen peroxidase in PBS for 10 min at RT. Antigen retrieval was carried out by boiling samples in Sodium Citrate (10 mM, pH 6) for 15 min. Sections were blocked using 5% Goat serum for 1 hour and incubated with the Click-IT EdU reaction cocktail following manufacturer’s protocol. Then, sections were washed with 1X TBST and re-blocked during 30 min before incubation with mouse anti-UHRF1 (1:50, sc373750, Santa Cruz Biotechnology) at 4 °C overnight. Detection was conducted with the M.O.M Immunodetection kit (BMK-2202 Vector laboratories) following manufacturer’s instructions and using Streptavidin Alexa Fluor-647 (1:200, S21374, Invitrogen) to detect UHRF1 staining. Finally, sections were counterstained with Hoechst and mounted for fluorescence microscopy.

The antibodies used for immunostaining, immunoprecipitation, and Western blotting analyses included the following: α-Tubulin mouse monoclonal antibody (T5168, Sigma), BrdU rat monoclonal antibody (ab6326, Abcam), Dnmt1 rabbit polyclonal antibodies for immunostaining and immunoprecipitation (BAM-70-201, Cosmo Bio) and only for immunoprecipitation (ab87656, Abcam), Flag mouse monoclonal antibody (F3165, Sigma), HA mouse monoclonal antibody (clone 12CA5, Roche), Histone H3 rabbit polyclonal antibody (ab1791, Abcam), Msh2 mouse monoclonal antibody (NA27, Calbiochem), Msh6 mouse monoclonal antibody (610919, BD Biosciences), Mlh1 mouse monoclonal antibody (554073, BD Biosciences), Myc mouse monoclonal antibody (05–9724, Millipore), Pms2 mouse monoclonal antibody (556415, BD Biosciences), Sox2 goat polyclonal antibody (sc-17320, Santa Cruz), and UHRF1 (Np95) mouse monoclonal antibody (sc-373750, Santa Cruz).

Protein extracts were probed with antibodies against human UHRF1 (sc-373750, Santa Cruz biotechnology, USA), KLF6 (sc-7158, Santa Cruz biotechnology, USA), COX-2 (A5787, ABclonal, USA), CSF1 (AF216, R&D Systems, USA), DNMT1 (sc-271729, Santa Cruz biotechnology, USA), and CCL14 (MAB3241, R&D Systems, USA) or β-actin (A5441, Sigma, USA). Stained blots were visualized with chemiluminescence assays using ECL detection reagents (Millipore, USA).