Antibodies against ha

293T cells were transfected with pCDNA-HA-BRCA2 plasmids(i.e. wild-type HA-BRCA2, germline c.407delA HA-BRCA2del407 and putative reversion c.402_413delTCTAAATTCTTG HA-BRCA2REV) for 72 hrs, and lysed in lysis buffer (0.5% NP40, 25 mM Tris pH 7.5, 450 mM NaCl, 0.5 mM EDTA and proteinase inhibitors). The cell lysates were then incubated with anti-HA agarose beads (Sigma). After three washes with wash buffer (0.5% NP40, 25 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA), the beads were boiled in SDS sampling buffer, followed by western blotting with antibodies against HA (Santa Cruz), PALB2 (Novus Biologicals) and RAD51 (Santa Cruz).

Yeast cells were grown in yeast/peptone media supplemented with 2% glucose or synthetic media as described previously described supplemented with 2% glucose and an amino acid mix consisting of histidine (2 mg/ml), leucine (3 mg/ml), lysine (3 mg/ml), tryptophan (2 mg/ml), adenine (2 mg/ml), uracil (2 mg/ml), methionine (1 mg/ml) at 100X (Lang et al., 2014 (link)), or amino acid drop-out mix lacking one or more nutrients to maintain plasmid selection. For experiments using Mup1, methionine was omitted. For experiments using Tat2, only histidine, leucine and lysine were added. Antibodies against HA were from Santa Cruz. Alexa-633 secondary antibodies were from Invitrogen (Carlsbad, CA). ADH1 antibody was from Sigma. For experiments with UL36, cells were treated with 0.1μM copper sulfate two hours prior to glucose starvation to induce the expression of UL36 fusion protein.