Journal: EMBO Reports

Article Title: SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

doi: 10.1038/s44319-024-00274-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: HEK293T cells were seeded on 10-cm 2 dishes, transfected with hSUN2-FLAG or INF2-CAAX separately using Lipofectamine 2000 (Thermo Fisher Scientific) according to the supplier’s protocol, and harvested by scraping into 1 mL PBS after 24 h. After a washing step with PBS at 3000× g for 5 min, the cells were lysed in Co-IP buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 1% glycerol, 1% NP-40, 0.05% SDS, 1× cOmplete protease inhibitor cocktail EDTA-free (Roche)) and the suspension left on ice for 30 min. Lysed cells were shortly sonicated (3×1 s, 25% power) and centrifuged for 15 min at 16,000× g . A sample of the supernatant was collected as “input” and the remaining solutions were either mixed or separately incubated with previously washed Anti-Flag M2 beads (Merck) by tumbling for 150 min at 4 °C.

Techniques: Recombinant, Sequencing, Negative Control, Transfection, Protease Inhibitor, Membrane, In Situ, Software, Microscopy, Laser-Scanning Microscopy