Co-immunoprecipitation of LSD1 Complex

In accordance with the protocol by Hattori [9 (link)], after the first stage of induction, shRNA-LSD1-927-treated hiPSCs were lysed with RIPA lysis buffer. Approximately 1000 μg of proteins was diluted with NET-gelatin buffer. Subsequently, rabbit anti-human LSD1, HDAC1, and CoREST antibodies (0.5–1 μg, Active Motif, USA) were added to the sample. An equal amount of normal rabbit serum was then added to the control group. After incubation with Agarose-A/G (sc2003, Santa Cruz, USA; 30 mL) on a 40 °C shaker overnight, precipitates were collected and washed three times. After the addition of 2× loading buffer and heat denaturation, proteins were separated on an SDS-PAGE gel. The primary antibodies used were as follows: mouse anti-human LSD1, HDAC1, and CoREST monoclonal antibody (1:200, Active Motif, USA). HRP-labeled horse anti-mouse IgG (1:200, L0807, Vector Laboratories, USA) was used for DAB staining.