Molecular Analysis of BRCA1 in Human Breast Cancer. Cells Under Oxidative Stress
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1 Moleculr Anlysis of BRCA1 in Humn Brest Cncer Cells Under Oxidtive Stress Brin L. Gilmore 1, Ynping Ling 1, Crly E. Winton 1,2, Ky Ptel 1, Vsile Krgeorge 1, A. Cmeron Vrno 1,3, Willim Dernley 1, Zhi Sheng 1,2,4, nd Deorh F. Kelly 1,2,4,5* 1 Virgini Tech Crilion Reserch Institute, Virgini Tech, Ronoke, VA 2 School of Biomedicl Engineering nd Science, Virgini Tech, Blcksurg, VA 3 Trnsltionl Biology, Medicine, nd Helth Grdute Progrm, Virgini Tech, Blcksurg VA 4 Virgini Tech Crilion School of Medicine, Virgini Tech, Ronoke, VA 5 Deprtment of Biologicl Sciences, Virgini Tech, Blcksurg, VA *Correspondence to: Deorh F. Kelly, Virgini Tech Crilion Reserch Institute, 2 Riverside Circle, Ronoke, VA Phone: ; Fx: ; Emil: dekelly@vt.edu Supplementry informtion 1
2 Supplementry Figures Ni-NTA elution profiles for HCC cells 50 IB: K48-Uq 50 Supplementry Figure 1. Western lot nlysis of Ni-NTA elution profiles for HCC cells. Western lot nlysis indictes wild type BRCA1 (~220 kd) eluted from Ni-NTA grose eds in the sme frction (E2) s BARD1 (~87 kd). Minor quntities of K48-linked uiquitin moieties (K48-Uq) were detected in the eluted mteril tht migrted t ~220 kd. Nucler extrct (); eluted mteril (E). 2
3 BRCA1-BARD1 Co-IP results for HCC cells Control cells, no oxidtive stress Input DEP IP* Cells experiencing oxidtive stress Input DEP IP* IB: Pser2 (control) Supplementry Figure 2. BRCA1 Co-IP experiments performed in HCC cells. () Co-IP experiments showed interctions etween BRCA1 (~220 kd) nd BARD1 (~87 kd) in the Ni-NTA elute in HCC control cells. () Co-IP experiments showed stle interctions etween BRCA1 (~220 kd) nd BARD1 (~87 kd) in the enriched nucler mteril of cells experiencing oxidtive stress. RNAP II phosphorylted t Pser2 repets (~260 kd) served s negtive control. Species-specific IgG control experiments showed low ckground signl.*denotes immunoprecipitted proteins (IP); unound / depleted mteril (DEP); immunolot (IB). 3
4 Ni-NTA Elution Profiles for HCC cells MW - (-) 2 H2O2 4 6 () 8 H2O10 2 MW FT (-) H2O2 IB: K48-Uq - c (-) H2O2 () H2O2 IB: K48-Uq () H2O Supplementry Figure 3. Ni-NTA elution profiles for HCC cell in the presence nd sence of tretement. () Western lot nlysis showed BRCA15382insC migrted t ~220 kd in untreted, () H2O2, HCC cells. BRCA15382insC migrted t ~2 kd in H2O2-treted cells, () H2O2. () K48-linked uiquitin moieties (K48-Uq) were detected t ~220 kd in untreted cells nd correspondingly t ~2 kd in H2O2-treted cells. (c) BARD1 (~87 kd) ws detected in untreted HCC cells in the sme frctions s BRCA15383insC, nd ws detected in H2O2-treted cells t ~87 kd. The elution profiles re more complex under oxidtive conditions. Nucler extrct (); Eluted mteril (E). 4
5 BRCA1 post-trnsltionl modifictions stress stress stress stress pbrca1 BRCA1 c stress stress d stress stress K48-Uq β-ctin Supplementry Figure 4. Post-trnsltionl modifictions detected in the nucler mteril of vrious rest cncer cells under oxidtive conditions. The nucler mteril of H2O2-treted nd untreted cells including HCC, HCCstress, nd HCC lines ws ssessed y western lot nlysis. () BRCA1 ws present in ech frction. () Phosphorylted BRCA1 (pbrca1) ws detected in HCC nd HCCstress cells with nd without H2O2 tretment using ntiodies ginst phosphorylted residue S1524. pbrca1 ws not detected in HCC cells. (c) K48-linked uiquitin moieties were elevted in ll cells upon H2O2 tretment especilly the HCC cells. (d) β-ctin served s loding control. 5
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