Dual color precision protein mw markers

Cells were treated as indicated in individual Fig. legends and whole cell extracts (WCE) were prepared in modified RIPA buffer (Sigma) with addition of protease and phosphatase inhibitors (Roche). Western analysis was performed and quantitated as described (Riggs et al., 2006 (link)). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Dual color precision protein MW markers (BioRad) were separated in parallel. Antibodies were purchased as follows: ERα (D-12, sc-8005), ERβ (sc-53494), Pdcd4 (sc-130545), from Santa Cruz Biotechnology (Santa Cruz, CA); AR (#3202) from Cell Signaling Technology; β-actin from Sigma; and α-tubulin (MS-581-P1) from Thermo Scientific/Lab Vision (Fremont, CA). Chemiluminescent bands were visualized on a Carestream Imager using Carestream Molecular Imaging software (New Haven, CT).

Cells were treated as indicated in individual figure legends and whole cell extracts (WCE) were prepared in the radioimmunoprecipitation assay (RIPA) buffer (Sigma) with addition of protease and phosphatase inhibitors (Roche). Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Dual color precision protein MW markers (BioRad) were separated in parallel. Antibodies were purchased as follows: cleaved-caspase-3 (ab214430), cleaved PARP (ab203467) and GAPDH (ab9657, Abcam) from Abcam; S100A8 (47310) and S100a9 (72590) from Cell Signaling; and Ltf (GTX81085) from GeneTex. The secondary antibodies conjugated to Fluors Alex-488 (A32723) or Alex-594 (A11012) were purchased from Invitrogen (Eugene, OR). The bands were visualized on the Odyssey Imager (LiCor Inc, Lincoln, NE).