α his

Whole cells or detergent-extracted cytoskeleton (CSK) samples were prepared as previously described [9 (link)] with a concentration of 1 × 10⁷ or 2 × 10⁷ (BSF or PCF) cells/well, as mentioned according to the experiments. Whole cell (T), CSK-enriched pellets (P), and soluble proteins (S) were prepared as described before [21 (link)], with the addition of 1% NP-40 and 150 mM NaCl. Proteins were separated on SDS-PAGE and transferred onto 0.2 μm PVDF membrane. The following primary antibodies were used in the indicated cases: α-BILBO1 (pAb 1:200), α-TbKINX1B (rabbit polyclonal 1:2000 or mouse monoclonal 1:1000), α-Tubulin (TAT1, 1:1000, [34 (link)]), anti-enolase (1:10,000), and α-His (Sigma H-1029, 1:3000). Incubations with secondary antibodies goat α-rabbit HRP (Jackson 115-055-068) and goat α-mouse HRP (Jackson 115-035-044) were also performed as cited above. Labelling was revealed with Clarity ECL Substrate (Bio-Rad) and revealed using ImageQuant LAS400. Quantifications were performed using ImageJ. Errors bars in graphs represent the standard error (n = 3).

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).