Sc 8005

The compounds tamoxifen, MKT-077, VER-155008, PU-H71 and HCQ were purchased from Selleck Chemicals (Houston, TX, USA). The compound S1g-2 was synthesized according to our previous report [17 (link)]. All the chemicals were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM. To obtain the final concentration, stock solutions were diluted in culture medium. Primary antibodies against LC3 (sc-398822), p62 (sc-28359), β-actin (sc-8432), Bim (sc-374358) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal antibody raised against a synthetic peptide antigen corresponding to the C-terminal of ERα36 was purchased from Abmart, Inc. (M000803, Shanghai, China). A mouse monoclonal antibody raised against amino acids 2-185 mapping at the N-terminus of ERα66 was purchased from Santa Cruz (sc-8005). To detect both ERα36 and ERα66 on the same gel, a previously reported antibody that can recognize either of ERα36 and ERα66 [22 (link)] was used (H222, Research Diagnostic). Antibodies against PARP (ab32138), and Bag3 (ab47124) were purchased from Abcam plc (Cambridge, MA, UK). Antibodies against EGFR (#4267), and Hsp70 (#4873) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Protein was prepared in NuPAGE Sample Buffer and Reducing Agent (Life Technologies, Carlsbad, CA, USA) using 10 μg (estrogen-related blots), 65 μg (V5 blot, T47D-ELF5-isoform 2-V5) or 25 μg (V5 blots, all other lines) per lane. Samples were separated on precast 15-well 4–12 % Bis-Tris (estrogen-related blots) or 10-well 10 % Bis-Tris (V5 blots) polyacrylamide gels (Life Technologies), transferred to polyvinylidene fluoride membrane, blocked in 5 % skim milk, and incubated overnight at 4 °C in primary antibody. Secondary horseradish peroxidase–conjugated antibody was added 1:2000 in 5 % skim milk (anti-mouse, NA931V, anti-rabbit, NA934V; GE Healthcare Life Sciences, Little Chalfont, UK). Proteins were detected using enhanced chemiluminescence solution (Western Lightning Plus; PerkinElmer, Waltham, MA, USA) and x-ray film (Fujifilm, Tokyo, Japan). Primary antibodies used were anti-V5 (sc-58052, 1:500–1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-transducin-like enhancer of split 1 (anti-TLE1) (ab183742, 1:1000; Abcam, Cambridge, UK), anti-ERα (sc-8005, 1:1000; Santa Cruz Biotechnology), anti-Forkhead box A1 (anti-FOXA1) (sc-101058, 1:1000, Santa Cruz Biotechnology), and anti-β-actin (AC-15, 1:20,000; Sigma-Aldrich, St. Louis, MO, USA).

MCF7 cells were plated on poly-l-lysine-coated cover slips (Fisher, Hampton, NH), while T47D, MDA-MB-231, and MDA-MB-468 cells were plated on Geltrex^®-coated cover slips (Invitrogen, Carlsbad, CA). Following treatment, the cells were fixed in 1% PFA for 10 min, washed twice with PBS, subsequently permeabilized in 0.3% NP-40/PBS for 10 min, and blocked in Image-iT FX signal enhancer solution (Invitrogen, Carlsbad, CA) for 30 min. Cells were incubated with ERα (1:50 dilution, SC-8005 Santa Cruz Biotechnology, Dallas, TX) and raptor (1:400, ab169506 Abcam, Cambridge, UK) primary antibodies in 1% BSA/PBS overnight at 4 °C. Cover slips were subsequently washed in PBS and incubated with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 goat anti-rabbit secondary antibodies (1:500 dilution, Invitrogen, Carlsbad, CA) for 1 h at room temperature in the dark. Following 5-min incubation with DAPI, cover slips were mounted using an Image-iT^® FX signal enhancer (Invitrogen, Carlsbad, CA) and imaged using a Nikon fluorescent microscope under ×40 magnification.