Mab7119

Proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific), and protein concentration was determined by bicinchoninic acid (BCA) protein assays (Thermo Fisher Scientific). Nuclear proteins were extracted using NE-PER™ nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Proteins (50 μg) were separated on 4%–20% gradient gels and transferred onto polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo transfer pack (Bio-Rad) and Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) and incubated with primary antibodies overnight at 4°C. The primary antibodies were SREBP2 (1:1,000, MAB7119, R&D Systems), Lamin A/C (1:500, SC-7292, Santa Cruz Biotechnology), HMGCR (1:1,000, MBS9406409, Mybiosource), and actin (1:1,000, SC-1616, Santa Cruz). Membranes were then incubated with IRDye 680CW or IRDye 800CW secondary antibodies (LI-COR Biosciences) for 1 h at room temperature. The membranes were scanned using the Odyssey infrared imaging system (LI-COR Biosciences).

Formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and matched lung metastasis samples from four individuals with breast cancer were selected. The institutional review board (IRB) of Cedars-Sinai Medical Center and the First Hospital of China Medical University (Shenyang, China) granted approval. The normal human lung tissue samples were purchased from US Biomax (T041a). Primary mouse breast tumors and lung metastasis samples (see Mouse models and study approval) were also used in the IHC assay. IHC staining was performed using the Vectastain ABC Kit (Vector Laboratories) and ImmPACT DAB Kit (Vector Labs). The primary antibodies were HMGCR (for human samples, 1:100, PA5-52547, Thermo Fisher Scientific), Hmgcr (for mouse samples, 1:70, MBS9406409, Mybiosource), HMGCS1 (for human and mouse samples, 1:100, GTX112346, GeneTex), CCR2 (1:100, NBP2-35334, Novus Biologicals), SREBP2 (1:100, MAB7119, R&D Systems), and Vimentin (1:100, sc-7557, Santa Cruz).