Milk powder

Cells were lysed in ice-cold RIPA buffer and protein concentrations were determined using the DC Protein Assay Kit (BioRad). Incubation with primary antibodies against ZEB1 (1:2000, Sigma #HPA027524), CHKα (1:500, Abcam #ab88053), TWIST1 (1:100, Santa Cruz #sc-81417), β-actin (1:1000, Santa Cruz #sc-130657) and α-tubulin (1:10000, Sigma #T9026) was performed overnight at 4°C on a 3D-shaker in 5% milk powder (Carl Roth) in TBST. As secondary antibodies we used goat-anti-rabbit IRDye800CW (1:10000, LI-COR #926-32211), goat-anti-mouse IRDye680RD (1:10000, LI-COR #926-68070) and goat anti-rabbit-HRP (1:10000, Jackson Immuno Research #111-035-144) diluted in blocking solution and incubated for 1 h at room temperature. Signal detection was performed either on a film based system by applying Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) or on a luminescence based system in a LI-COR Odyssey CLx Imager (LI-COR). Densitometry was done using supplied software from LI-COR or ImageJ software [75 (link)]. Densitometry values for ZEB1 and CHKα were normalized to the corresponding alpha tubulin values, TWIST1 was normalized to beta actin.

KDM5D Knockdown (KD) and Wildtype (WT) ITSCs were harvested by surface scraping followed by cell lysis on ice with lysis buffer (10 mM Tris, 1% SDS, 3 mM EDTA). Protein concentrations of the samples were adjusted to 20 µg total protein amount and mixed with 4× loading buffer followed by heating at 95 °C for 8 min. Samples were subjected to electrophoresis on 10% denaturing SDS polyacrylamide gel and transferred with a semi-dry blotter to a nitrocellulose membrane (Carl Roth GmbH, Karlsruhe, Germany). Blocking of membrane with 5% milk powder (Carl Roth GmbH, Karlsruhe, Germany) in 1× TBS with 0.05% Tween 20 (Sigma-Aldrich, Taufkirchen, Germany) was followed by incubation with the first antibody against JARID1D/KDM5D (1:500; Cell Signaling Technology Inc., Danvers, MA, USA) in 1× TBS with 5% milk powder and 0.05% Tween 20 while shaking at 4 °C overnight. HRP-linked secondary antibody (1:4000; Cell Signaling Technology Inc.) was applied for 1h at RT. Visualization was performed via enhanced chemiluminescence. Beta-actin antibody (1:2000; Cell Signaling Technology Inc.) was applied as control for 1 h at RT followed by exposure to secondary antibody as described above.

In brief, viral protein was coated into 96-well Nunc MicroWell^TM plates (Thermo Fisher, US) by diluting the stock to 0.75 µg/mL in NaCO₃ buffer (0.1 M, PH 9.6) and coating with 50 µL per well with overnight incubation at 4 °C. The plates were washed 5 times with 0.1% PBS-T then blocked with 5% milk powder (Carl Roth, Germany) in PBS-T for one hour at room temperature and the wash repeated. Serum to be tested were diluted 1:200 in 1% milk powder in PBS-T. After one-hour incubation at room temperature, plates were washed 5 times with PBS-T and goat anti-human antibody labelled with Horseradish peroxidase enzyme (Dianova GmbH, Hamburg, Germany) at a dilution of 1:2000 was added. For livestock testing, HRP-coupled donkey anti-sheep, goat anti-swine, and donkey anti-goat antibodies (Dianova GmbH, Hamburg, Germany) were used for sheep, swine, and goat testing, respectively, also at a 1:2000 dilution. The conjugate was incubated at room temperature for 30 min after which plates were washed 5 times with PBS-T and an enzyme substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB) (Mikrogen Diagnostik, Neuried, Germany) was then added. This was kept in the dark for 15 min and stopped with 2 Molar sulphuric acid (H₂SO₄) and the absorbances read at 450 nm and 630 nm on a Biotek synergy 2 (BioTek Instruments Inc., Winooski, VT, USA) multi-detection plate reader.