The MDAMB231, MDAMB468, MCF7, T47D, ZR75-1, OVCAR3, and SKOV3 cell lines were obtained from the American Type Culture Collection (Manassas, VA). Protein lysates of 52 breast cancer cell lines were prepared as previously described [29 (link)]. The human tumor sets used herein were obtained using Institutional Review Board-approved protocols and are as follows:

Set A (128 tumors): For comparison of RPPA with transcriptional profiling (e.g., for protein–mRNA correlations), 128 stored primary breast tumors were obtained from patients treated in the Danish DBCG82 b and c studies [45 (link)] (Table 2).

Set B (ten tumors): For the studies of intratumoral heterogeneity and total and phosphoprotein stability, a prospective study was undertaken to collect primary breast tissue at breast surgery in ten patients with breast cancer under an Institutional Review Board (IRB)-approved protocol. Each tumor was sectioned with assistance from a breast pathologist and immediately snap frozen (three pieces) or left at room temperature in closed eppendorf tubes without any added buffer for 0.5/1/2/4/6/24 h (1 piece/time point) prior to freezing (−85°C). Protein was extracted from each piece of tumor without thawing.

Set C (95 tumors): Ninety-five stored primary breast tumors were obtained from the breast tumor frozen tissue bank at M. D. Anderson Cancer Center under an IRB-approved protocol (Table 2). Protein was extracted from these 95 tumors, including from two independent sections (“biologic replicates”) derived from 49 of the 95 tumors.

Note that Table 2 does not show the clinical data for Set B since the clinical data for this set were not utilized in this study.
MDAMB231 and MDAMB435 breast cancer xenografts were assessed for total and phosphoprotein stability using the same approach as with human tumor set B above. After animal sacrifice, the xenograft tumors were sectioned and immediately snap frozen or left at room temperature in closed eppendorf tubes without any added buffer for 0.5/1/ 2/4/6 h (1 piece/time point) prior to freezing (−85°C). As with the human tumors, protein was extracted from each piece of tumor without thawing.