Ne per nuclear and cytoplasmic extraction reagents kit

Nuclear extracts were isolated following the instruction of NE-PER Nuclear And Cytoplasmic Extraction Reagents Kit (Pierce-Thermofisher Scientific). Cells were washed by suspending the cell pellet with phosphate-buffered saline. We added the CER I buffer and CER II buffer to swell the cells on ice for 10 min and then vortexed for 10 s. Subsequently, samples were centrifuged for 10 s and the supernatant fraction was discarded. Pellets were resuspended in NER buffer and incubated on ice for 40 min for high-salt extraction. Cellular debris was removed by centrifugation for 10 min at 16,000 g and the supernatant fraction (containing DNA-binding proteins) was stored at −80 °C until use. Protein concentrations were determined by the BCA Protein Assay Kit (Bio-Rad, Hercules, CA) [36 (link)].

Cytoplasmic and nuclear fractions were isolated from ~ 10–20 × 10⁶ Treg cells from IL-2/media only cultures or Tregs from IL-2/media MSC co-cultures using the NE-PER Nuclear and Cytoplasmic extraction reagents kit (Invitrogen 78833) according to the manufacturer's instructions. In brief, Treg cells were removed from co-culture, washed with PBS, and ice cold CER1 reagent was added, and samples were pelleted by centrifugation. Samples were vortexed for 15 s and incubated on ice for 10 min. Ice cold CERII reagent was added and samples were vortexed for an additional 5 s and incubated on ice for 1 min. After additional 5 s vortex, samples were centrifuged for 5 min at maximum speed. The supernatant containing the cytoplasmic extract was transferred to a new tube. The insoluble pellet fraction containing the nuclei was suspended in ice-cold NER and vortexed for 15 s every 10 min for 40 min. Nuclear fraction was centrifuged at maximum speed for 10 min and supernatant containing the nuclear extract was transferred to a new tube. Isolated fractions were kept on ice. Protein concentration was determined by Bradford assay and equivalent amount of total protein for each sample (5–10 μg nuclear fraction, 20–40 μg cytoplasmic fraction) was loaded onto a 4–20% SDS-PAGE gel and processed for analysis of BACH2 and SENP3 expression by western blotting as described above.

MCF7 Tet-On cells grown in hormone-depleted medium for a day were transiently transfected with pcDNA HA-ERα WT or mutant plasmids. A day after transfection, cells were exposed to either 10 nM E2 or vehicle (DMSO) control for 24 hours and harvested. Nuclear and cytoplasmic components were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, catalog 78833) following the manufacturer-recommended protocols. Ten to fifteen percent (vol/vol) of the cytoplasmic and nuclear fractions were loaded onto a NuPAGE 4%–12% Bis-Tris gel, and standard immunoblotting techniques were used to determine the levels of HA-tagged ERα, HDAC2, and GAPDH. HDAC2 was used as a loading control for the nuclear fraction (44 (link)) and GAPDH for the cytoplasmic fraction (45 (link)). Image Studio Lite 5.2 was used to quantify the intensity of the bands seen, and the relative enrichment in the nucleus as compared with the cytoplasm was calculated as (Nuclear HA–ERα/nuclear HDAC2)/(Cytoplasmic HA–ERα/cytoplasmic GAPDH) and plotted in Microsoft Excel.

Subcellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, #78835) according to the manufacturer’s instructions. Detailed information of total protein extraction, immunoblotting, and densitometric quantification (Supplementary Figs. S7, S8, and Table S2) are described in the Supplementary Materials and methods.

Cells or tissues were washed with cold PBS and lysed in RIPA buffer containing 1% proteinase inhibitor cocktail solution (Sigma-Aldrich) on ice for 30 min. The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, USA) was applied to isolate nuclear fractions. Lysates were centrifuged, and protein was quantified using the BCA assay kit (Beyotime, Jiangsu, China). SDS-PAGE electrophoresis was performed on proteins from lysates and transferred them to PVDF membranes (Millipore, Bedford, MA). Membranes were incubated with primary antibody overnight at 4 °C, and then detected by Enhanced Chemiluminescence Detection Kit (BOSTER, USA). Lamin B1 was used as an endogenous control of cell nuclear fraction. All antibodies used were shown in Additional file 2: Table S2.