Western blocking buffer

Cells were differentiated into mature brown adipocytes according to a standard adipogenic differentiation protocol. After that, cells were scraped from tissue-culture plates into RIPA buffer (Boston BioProducts Inc., Ashland, MA, USA) supplemented with protease and proteinase inhibitors cocktails (Sigma-Aldrich, Dallas, TX, USA) and further homogenized for protein detection. Protein concentrations were determined using a Pierce BCA kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. For immunoblots, lysates were diluted into Laemmli buffer and boiled for loading onto 10% Tris gels for SDS–PAGE. After complete separation of the proteins, they were transferred onto a PVDF membrane (Amersham Biosciences, Amersham, UK), blocked in western blocking buffer (Roche, Basel, Switzerland), and primary antibodies were applied in blocking buffer overnight at 4 °C. After washing 4× for 15 min with TBS-T, secondary antibodies were applied for 1 h in blocking buffer. Membranes were washed again 3× times for 15 min in TBS-T and developed using chemiluminescence (Thermo Fisher, Waltham, MA, USA).

Immunostaining was performed after FISH development by reblocking planarians in Blocking Solution (5% heat inactivated horse serum, 5% Roche Western Blocking Buffer in TNTx) for 2 hours at room temperature, labeling mitotic cells with anti-phospho-Histone H3 (Ser10) (1:2,000) in Blocking Solution overnight at 12°C, washing 6X in PBSTx (30 minutes each), reblocking for 2 hours at room temperature, and incubating with HRP-conjugated goat anti-mouse (1:500) in blocking solution overnight at 12°C. Planarians were washed 6X in PBSTx (30 minutes each) and TSA was performed for 30 minutes.