D3669

HeLa.Scramble, HeLa.shUHRF1 expressing Flag-tagged UHRF1 or HeLa.shFANCD2 expressing Flag-tagged FANCD2 cells were treated with TMP/UVA as described. Cell pellets were incubated with Buffer A (0.1% Triton X-100, 20mM Hepes pH7.9, 5mM MgCl₂, 10% Glycerol, 1unit/μl Benzonase and 2.5mg/ml DSP (D3669, Sigma)) for 30 minutes on ice, and the Tris pH 8.0 was added to the mixture to 0.2M to quench DSP. 10 times pellet volume of Buffer B (0.15% Triton X-100, 20mM Tris-HCl pH 8.0, 5mM MgCl₂, 10% Glycerol, 300mM KCl and 0.2mM PMSF) was added to the mixture and incubated for 10 minutes for extraction. Lysates were clarified by centrifugation, and supernatant was used for immunoprecipitation. M2 agarose beads was added to the lysates, and incubated for 2 hours. The resin was washed extensively, and eluted with 0.5mg/ml Flag peptide.

Protein cross linking was performed using 2mM DSP (D3669; Sigma) using a protocol previously described [25 (link)]. Co-IP was performed as follows: 1mg of total protein incubated with anti-V5 antibody-coated agarose beads (A7345; Sigma) overnight at 4°C followed by six washes with cold protein extraction buffer. Elution was performed by incubating the beads with 2x Western blot sample buffer at 65°C for 20 minutes. Samples to detect proteins were run on pre-made gels (Novex, Life Sciences) and transferred onto PVDF membranes. Polyclonal antibodies were used for FRQ (1:250), WC-1 (1:250) and WC-2 (1:5000) and commercial monoclonal V5 antibody (1:5000, Invitrogen) was used for VVD. Goat Anti-Rabbit HRP conjugate and Goat Anti-Mouse HRP conjugate (BioRad) were used a secondary antibodies. Membranes for anti-V5 IP were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and the signal was captured using X-ray film (GE Healthcare). Western Blots were quantified using NIH ImageJ software.