Ab155296

RIPA lysis buffer (Beyotime) was applied for extracting the total protein. After measurement of protein concentration, protein (about 40 µg/lane) was subjected to SDS-PAGE (Beyotime). Subsequently, the proteins were transferred to nitrocellulose membranes (Invitrogen), which were then blocked with 5% non-fat milk (Yili, Beijing, China) and incubated with primary antibody overnight at 4°C. The primary antibodies including cluster of differentiation 63 (CD63; 1:1000, ab118307), cluster of differentiation 9 (CD9; 1:500, ab223052), TPD52 (1:5000, ab155296), and GAPDH (1:2000, ab37168) were bought from Abcam (Cambridge, UK). After that, the corresponding secondary antibody (1:4000, ab205718, Abcam) was used for the combination with the primary antibody. At last, the visualization of protein blots was achieved by an enhanced chemiluminescence reagent (Solarbio, Beijing, China).

Transfected cells were lysed using RIPA lysis buffer (Thermo Fisher, Wilmington, DE, USA) containing protease inhibitors (Beyotime, Shanghai, China) to extract the total protein. After quantification by using bicinchoninic acid (BCA) protein assay kit (Tanon, Shanghai, China), protein samples (about 30 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the gels were transferred onto the polyvinylidene fluoride (PVDF; Beyotime) membranes. Then, 5% non-fat milk (Sangon Biotech) in PBS containing 0.1% Tween 20 (PBST) was used to block the membranes, and then the membranes were immunoblotted for 12 h at 4°C by primary antibodies against TPD52 (1:5000, ab155296, Abcam, Cambridge, UK) or β-actin (1:2000, ab8227, Abcam). Following washing with PBST, membranes were incubated by secondary antibodies (1:4000, D110058, Sangon Biotech). Immunoreactive bands were examined by enhanced chemiluminescence (ECL) reagent (Tanon). Protein expression of TPD52 was evaluated using ImageJ software and normalized to the level of β-actin.

Cell lysates containing equal amounts of proteins underwent 8% SDS-polyacrylamide gel electrophoresis followed by transfer onto PVDF membranes. After blocking non-specific binding sites with 5% dried skimmed milk solution for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies (TPD52, 1:10,000 dilution, ab155296, Abcam; DNAJB1, 1:500 dilution, SAB2100604, Sigma-Aldrich; TACC3, 1:500 dilution, ab138262, Abcam; Ephrin A1, 1:500 dilution, ab199697, Abcam; β-actin, 1:1,000 dilution, ab227387, Abcam). The membranes were then incubated with secondary antibodies conjugated to horseradish peroxidase (against mouse, 1:10,000 dilution, A9917, Sigma-Aldrich; against rabbit, 1:5,000 dilution, A9169, Sigma-Aldrich) for 1 h at room temperature and the signals were finally detected with ECL system. β-actin was used as a loading control, and the data were quantitatively measured using ImageJ software (version 1.48; National Institutes of Health).