Dmem cell culture media

HEK293 cells from ATCC (ATCC# CRL-1573) were cultured in DMEM cell culture media (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1x penicillin-streptomycin (Life Technologies). Media change was performed every other day until the cells reached 80% confluency, after which they were transferred to new culture dishes. Transfection of the HEK293 cells with exogenous DNA (1 μg) was accomplished using Lipofectamine 2000 as per the manufacturer’s instructions. Transfection took place when the cells reached 50–60% confluency in six-well culture plates. Co-IPs were conducted as below mentioned.

Picoline borane, DMEM cell culture media, and phosphate buffered saline (PBS, pH 7.4) were from Sigma-Aldrich (St Louis, MO). Polyethylene-glycol was purchased from Merck (Kenilworth, NJ). Fetal bovine serum, trypsin-EDTA, and 3-aminopropyltriethoxysilane (APTES) were from Thermo Fisher Scientific (Waltham, MA). Glutaraldehyde was purchased from Carl Roth Chemicals (Karlsruhe, Germany). Methanol and absolute ethanol were purchased from VWR (Radnor, PA). Hydrochloric acid, sulfuric acid, and dry toluene were purchased from Molar Chemicals Kft. (Halasztelek, Hungary). Kanamycin was from SERVA Electrophoresis GmbH (Heidelberg, Germany). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Biosynth AG (Staad, Switzerland). The SDS PAGE gel was made from 40% acrylamide solution from Bio-Rad (Hercules, CA) and HPLC grade water in a ratio of 37.5:1. Buffer “A” contained NaH₂PO₄ from Merck (Kenilworth, NJ) and NaCl from VWR (Radnor, PA). Imidazole for buffer “B” was purchased from Merck.

Skin biopsies or feather pulp samples were collected from one individual per species: from animals captured in their natural environment (H. torquata and T. s. surrucura) or from ex situ individuals (P. brasilianus, P. inscriptus, and R. t. tucanus) (Table 1). We performed fibroblast cell culture to obtain chromosome preparations. Cells were cultured in flasks (25 cm²) with DMEM cell culture media (Dulbecco’s Modified Eagle’s medium, Sigma-Aldrich, MO, USA), supplemented with 20% fetal bovine serum (GIBCO/Thermo Fisher Scientific, USA), 1% penicillin (10,000 units/mL)–streptomycin (10,000 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C [31 (link)]. Metaphase chromosomes were obtained according to standard procedures involving colcemid exposure (1 h, 37 °C), hypotonic treatment (0.075 M KCl, 15 min, 37 °C) and fixation with methanol:acetic acid (3:1).