Las4000 imager

Proteins were separated and detected as previously described [28 (link)]. In brief, SDS-PAGE gel (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) was used to separate proteins and then transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Blocking of membranes was achieved by using 5% dry milk dissolved in Tris Buffer Saline with 1% Tween 20 (TBST) and the following antibodies were incubated overnight at 4 °C: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-NOXA (CST14766, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-phospho-ERK1/2 (CST4376, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling). Anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) was used and immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When required, immunoblots were stripped in 0.1 M glycine pH 2,5, 2% SDS for 40 min and washed in TBS. The visualization of the bands was done using the LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to quantify the integrated optical density of bands.

All the gel and dot blot pictures have been acquired on a LAS 4000 imager (GE). Gel bands were quantified on the raw picture with ImageJ software and data was plotted with Graphpad prism software. Microscopy was performed on a Nikon Eclipse Ti inverted fluorescence microscope using a CFI plan apochromat Lambda 40× air objective. Cells were imaged in widefield fluorescence mode using lasers with wavelengths 405 nm (300 mW) and 488 nm (200 mW). Fluorescence emission was detected using ET450/50m and ET525/50m filters and an EMCCD camera: Andor iXon Ultra 888 (1024 × 1024 sensor size, 13 μm pixel size).

Cell lysate was lysed with RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM ethlenediaminetetraacetic acid, 1% NP-40, 0.1% sodium dodecyl sulfate). Nuclear isolation and mitochondrial isolation were performed using the nuclear isolation kit and mitochondrial isolation kit for cell and for tissue (Thermo Scientific, Rockford, IL, USA), respectively, according to the manufacturer’s instructions. Total protein concentrations were quantified by a colorimetric detection assay (BCA Protein Assay, Pierce, USA). Equal amounts of protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). Interested proteins were probed by primary antibodies and corresponding peroxidase-conjugated secondary antibodies, followed by detection by ECL (Merck Millipore Corporation, Darmstadt, Germany) and capture using the LAS-4000 imager (GE Healthcare Biosciences, Pittsburgh, PA).

2%
agarose (Sigma-Aldrich)
gels were cast in 0.5× TBE buffer (VWR) supplemented with 10
mM MgCl₂ and 0.5 mg/mL ethidium bromide (Sigma-Aldrich).
For all samples, gels were run in 0.5× TBE buffer supplemented
with 10 mM MgCl₂ at 90 V for 3 h on ice. Gels were imaged
under a GE LAS 4000 imager.

Tissues and cells were homogenized in Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Homogenates (20 μg of total protein) were separated by SDS‐PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against Sirt6, GATA‐1, Ac‐K, Ac‐H3K27 (Cell Signaling, Beverly, MA, USA), PGC1α, PRDM16, CCR3, IL‐4Rα (Abcam), α‐tubulin, UCP1 (Sigma‐Aldrich), myc, HA tag (Thermo Fisher Scientific, Waltham, MA, USA), Ac‐H3K27 (Active Motif, Carlsbad, CA, USA), Ym1 (Stemcell Technologies, Cologne, Germany), and arginase 1 (Arg1, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive bands were detected with a Las‐4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).