Agarose a g

3T3-L1 cells were transiently transfected with the NEK8 overexpression plasmid with 0, 0.5, 1, and 2 μg, and then incubated for an additional 48 hours. Then, 3T3-L1 cells were collected and lysed in RIPA lysis buffer (Yeasen, Shanghai) supplemented with protease inhibitor cocktail (Yeasen) for 30 min at 4°C, centrifuge at 8, 800 rpm for 10 min, and the supernatant was collected and divided into three 1.5 ml tube (one for input, one for IgG, and one for IP). Immunoprecipitation was conducted using antibodies against NEK8 or TAZ. Briefly, 1 μg primary antibody against NEK8 or TAZ was added to 200 μL cell lysates and incubated with rotation at 4°C for 2 h. Rabbit immunoglobulin G (IgG) was used as a negative control. Subsequently, proteins were precipitated with agarose A/G (Santa Cruz) and washed 5-6 times with RIPA lysis buffer. Immunoprecipitates and input were subjected to run SDS-PAGE, the next step same as western blot assay.

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.