Prbser807

The following primary antibodies were used in this study for immunoblotting: pRB^ser780 (CST-3590), pRB^ser807 (CST-8516), total-RB (CST-9309), cyclin D1 (CST-2922), cyclin D3 (CST-2936), pAKT^ser473 (CST-9271), pAKT^Thr308 (CST-9275), total-AKT (CST-9272), pEGFR^Tyr1068 (CST-3777), total-EGFR (CST-2232), pERBB2^Tyr1248 (CST-2243), total-ERBB2 (CST-4290), pERBB3^Tyr1222 (CST-4784), pIGF1R^Tyr1135 (CST-3918), pS6K^Ser235/236 (CST-2211), total-S6K (CST-2217), Raptor (CST-2280), RheB (CST-13879), p4EBP1^Thr37/46 (CST-2855), 4EBP1 (CST-9452), pSIN1^Thr86 (CST-14716), SIN1 (CST-12860), pER^ser167 (CST-5587), Rictor (CST-2114) and Deptor (SCT-11816) were purchased from Cell Signaling Technology. p107 (sc-318), p130 (sc-317), total-ER (sc-8002, F-10), ERBB3 (sc-415) and IGF1R (sc-713) were purchased from Santa Cruz Biotechnology. β-tubulin (T-9026) were from Sigma-Aldrich and Ki67 from Clinisciences. The following antibodies were used for immunohistochemistry: pERK1/2^Thr202/4 (CST-4370), pAKT^ser473 (CST-4060), pS6K^Ser235/6 (CST-4858), pmTOR^Ser2448 (CST-2976) and p4EBP1^Thr37/46 (CST-2855) were purchased from Cell Signaling Technology. Ki67 was purchased from Clinisciences. Reagents were obtained from the following sources: 17-β-oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) from Sigma-Aldrich, fulvestrant from Tocris, and neratinib and vistusertib from SelleckChem.

The PDX tumors were collected, fixed with 10% formalin for 24 hours, washed with PBS and maintained in 70% ethanol at 4°C. The tissue samples were dehydrated in graded ethanol, xylene, and embedded in paraffin. Immunohistochemistry of paraffin embedded section (5 µm) was performed using DAKO Envision+System (Dako, Santa Clara, CA). The following primary antibodies were used: PAX8 (Proteintech, Rosemont, IL, #10336-1-AP, 1:1000), ARID1A (Sigma-Aldrich, #HPA005456, 1:500), napsinA (Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UK, #NCL-L-NapsinA, 1:400), racemase (Zeta Corporation, Arcadia, CA, # Z2001L, 1:50), Ki67 (Dako, #M7240, 1:100), p-Rb (Ser807/811) (Cell Signaling, #8516, 1:200), γH2AX (Ser139) (Cell Signaling, #9718, 1:500), and cleaved caspase-3 (Asp175) (Cell Signaling, #9664, 1:200). Each antibody was incubated for 40 minutes at room temperature. Antigen retrieval for all targets was performed using a pressure cooker in citrate buffer (pH = 6). Appropriate positive and negative (incubation with secondary antibody only) controls were stained in parallel for each round of staining. The percentage of positive cells and staining intensity were reviewed by pathologist and quantified by ImageJ.