T6199 clone dm1a

Samples were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 4% SDS) and subjected to iterative rounds of heating at 95 °C and cup horn sonication until lysis was complete. Protein concentration was determined by BCA assay, and equal amounts of proteins were loaded on a 10% Tris-glycine SDS-polyacrylamide gel that was run at 130 V. Proteins were transferred overnight at 4 °C at 30 V onto a polyvinylidene fluoride (PVDF) membrane. Membranes were cut and blocked for 1 h in blocking buffer (5% milk, 0.2% Tween-20 in PBS). Membranes were incubated with primary antibodies in block for 3 h at room temperature: ACAT1 (1:1000, PA5-82154, Thermo Fisher Scientific), IE1 (1:500, clone IB12, gift from Thomas Shenk), pUL26 (1:200, gift from Thomas Shenk), pUL99 (1:200, clone 10B4, gift from Thomas Shenk), and tubulin (1:5000, T6199 clone DM1A, Sigma). Membranes were incubated for 1 h with secondary antibodies in block: Alexa 680 and 800 anti-mouse or anti-rabbit IgG (H+L) (Invitrogen, 1:10,000). To determine knockdown efficiency, densitometry analysis was performed using the ImageStudioLite for Odyssey. Intensities of ACAT1 were normalized to those of tubulin.

Cells were lysed in 2% SDS, 1% NP-40, 50 mM Tris pH 7.5, and 150 mM NaCl. Protein-lysates were equalized based on protein concentration measured using the BCA Protein Assay Reagent (Thermo Fisher Scientific). Protein samples were separated on Novex 4–12% Bis-Tris gradient gels using MOPS SDS running buffer and NuPage LDS sample buffer (all Thermo Fisher Scientific); subsequently, proteins were transferred onto Immobilon‐FL membranes (Merck Millipore). The following primary antibodies were used: Anti-PARP1 (rabbit polyclonal, Cell Signalling Technology #9542, 1:1000), anti-OBFC1 (STN1, mouse monoclonal, Santa Cruz sc-376450, 1:1000), anti-53BP1 (rabbit polyclonal, NOVUS biologicals NB100-304, 1:1000), anti-Mad2L2/REV7 (rabbit monoclonal, Abcam ab180579, 1:1000), anti-Cas9 (mouse monoclonal, 7A9‐3A3 Cell Signaling Technology, 1:1000) and anti-Tubulin (mouse monoclonal, Sigma-Aldrich, T6199 clone DM1A, 1:5000). The secondary antibodies CF680 goat anti‐rabbit IgG and CF770 goat anti‐mouse IgG (Biotium, both at 1:10.000) and the Odyssey CLx infrared imaging scanning system (LI‐COR Biosciences) were used to detect protein expression.