Abraxas

Protein concentrations were determined using the BCA Protein Assay Kit (23225, Thermo Fisher Scientific). The following antibodies were used: BRCC3: ab108411, Abcam, Cambridge, UK; Tubulin: T5168, Sigma-Aldrich; ABRO1: ab74333, Abcam; Abraxas: ab139191, Abcam; BABAM1: sc-160990, Santa Cruz, Dallas, TX, USA; UIMC1: 14466S, New England Biolabs, Ipswich, MA, USA; FLAG-tag (DYKDDDDK): 2368, Cell Signaling Technology, Danvers, MA, USA; HA-tag (YPYDVPDYA): 901533, BioLegend; G-CSF: sc-53292, Santa Cruz; β-Actin HRP: ab20272, Abcam; secondary anti-mouse HRP: 7076S, Cell Signaling; secondary anti-rabbit HRP: 7074S, Cell Signaling.

For testing protein-protein interaction, BRCA1 expression constructs were transfected into 293T cells plated at a density of 5 × 10⁵ cells per well in six-well plates and transfected with 2 μg of plasmid per well using FuGENE HD or X-tremeGENE 9 XP. Cells were collected 30 hr after transfection and lysed with 350 µl of NETNG-300 (300 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 0.5% Nonidet P-40% and 10% Glycerol) containing Complete protease inhibitor cocktail (Roche). The 3xMyc-tagged BRCA1 proteins were IPed for 3–4 hr with anti-Myc (9E10, Covance) and protein A-agarose beads (Roche). For western blotting analyses, proteins were resolved on 4–12% Tris-Glycine SDS gels, transferred onto nitrocellulose membranes and probed with the following antibodies- Myc (9E10, Covance), PALB2 (M11) (Xia et al., 2006 (link)), BARD1 (H300, Santa Cruz), BRIP1 (a gift from Dr. Sharon Cantor, University of Massachusetts Medical School), Abraxas (ab139191, AbCam). The secondary antibodies used were Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (NA931V, GE Healthcare) and donkey anti-rabbit IgG (NA9340V, GE Healthcare). Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used to develop the blots.