Immunoprecipitation and Immunoblotting of Lem27

HEK293T cells were resuspended with 1 ml NP40 lysis buffer for 10 min on ice, and the lysates were then centrifugated at 12,000xg at 4°C for 10 min. Beads coated with Flag- or HA-specific antibody were added to cleared lysates and incubated on a rotatory shaker for 8 hr at 4°C. After washed three times with the NP40 lysis buffer, aliquots of beads were mixed with the purified Lem27 or Lem27_C24A in 20 μL DUB buffer at 37°C. At the indicated time points, reactions were stopped by adding 5 μL 5 × SDS loading buffer and were heated at 95°C for 5 min. After SDS-PAGE, proteins were transferred onto nitrocellulose membranes (Pall Life Sciences) for immunoblotting after being blocked in 5% nonfat milk in PBST buffer for 1 hr. Primary antibodies used in this study and their dilutions are as follows: α-Flag(Sigma, Cat# F1804, 1: 3000), α-GFP(Sigma, cat# G7781, 1:5000), α-His(Sigma, cat# H1029, 1: 10,000), α-HA (Santa Cruz, cat# sc-7392, 1: 1000), α-ICDH (1: 20,000) (Xu et al., 2010 (link)), α-tubulin (DSHB, E7, 1: 10,000). Antibodies specific for Lem27 were generated by immunization of rabbits with purified His₆-Lem27 using a standard procedure (AbMax Biotechnology Co., LTD, Beijing, China) and were used at 1:500. Washed membranes were incubated with appropriate IRDye secondary antibodies and signals were detected and analyzed by an Odyssey CLx system (LI-COR).