Inpp4b

Cells were lysed in Cell Signaling lysis buffer. Western blotting was performed with the following antibodies: Cell Signaling: INPP4B (#8450), phospho-Akt (Ser473) (#4060), Erk1/2 (#4695); Santa Cruz: PTEN (sc-6817-R), Akt 1/2 (sc-1619), BRCA1 (sc-642); Millipore: H2A.X (Ser139) (#05–636), ATM (#07–1286); Bethyl: ATR (A300–138A).

BCa cells were lysed for Western blotting and proteins were detected using the antibodies, GAPDH (Santa Cruz 32233), P-Ser473 AKT (Cell Signaling 4060), INPP4B (Santa Cruz 12318), Total AKT (Cell Signaling 9272), ERα (produced in our lab, SC1-1), and p27 (Santa Cruz 528). Tissue fixation and sectioning were processed as previously described [36 (link), 73 (link)]. Tissue sections were incubated with ERα antibodies (Santa Cruz, MC-20), P-Ser473 AKT (Cell Signaling 4060), INPP4B (Abcam EPR3108), Ki67 (Novocastra).

Cell proteins were extracted with buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X 100), supplemented with protease and phosphatase inhibitors (GeneDepot, Barker, TX). For each sample, 50 μg of protein was resolved on SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed using the following antibodies: INPP4B 1:1000 (Santa Cruz), FLAG M2 (1:5000) (Sigma-Aldrich), β-Tubulin (1:2000) (Millipore, Billerica, MA), total Akt (1:1000), phospho- S473 Akt (1:1000), COX-2 (1:1000), phospho-S235/236 S6 (1:1000), total S6 (1:1000), survivn/BIRC5 (1:1000), pan phospho-PKC (1:1000) (Cell Signaling Technology, Beverly, MA), PAK6 (1:400) (R&D Systems, Minneapolis, MN). Luminescent signal was captured on a Gel Logic 2000 imaging system with Carestream Molecular Imaging software (Carestream, Rochester, NY).