Ab183711

For LSD1 interaction studies with CoREST and HDAC2, total protein lysates were prepared from Caco-2 cells (1 × 10⁷) using RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1× protease inhibitor cocktail). Cells were lysed for 30 min at 4 °C prior to LSD1 pulldown with 15 µg of rabbit polyclonal anti-KDM1/LSD1 (ab17721, Abcam) and Protein A magnetic beads (16-661, Merck Millipore). The beads were washed, and the bound proteins were eluted with immunoblot loading buffer containing beta-mercaptoethanol, followed by incubating at 95 °C for 5 min. Samples were subjected to western blotting as described above. Membranes were probed with rabbit anti-CoREST antibody (ab183711, Abcam) or rabbit anti-HDAC antibody (57156, Cell Signaling Technology) overnight at 4 °C, and detected using mouse and rabbit HRP antibodies and enhanced chemiluminescence reagents. Signals were imaged with the iBrightTM CL1500 Imaging System.
For ACE2 methylation studies, cell surface protein extracts were prepared from Caco-2 cells (1 × 10⁷) using the PierceTM Cell Surface Protein Biotinylation and Isolation Kit as described above. Membrane extracts were subjected to ACE2 pulldown with 15 µg rabbit anti-ACE2 antibody (SN0754) (NBP2-67692, Novus Biologicals) as described above, and western blot performed using the rabbit anti-pan methyl Lysine antibody (ab7315, Abcam).