Ab105381

Western blot was performed as previously described [42 (link)]. Briefly, cells were lysed in the radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) Sodium deoxycholate, 0.1% (w/v) SDS] with protease inhibitors cocktail (Sigma) added freshly. The lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore), which were blocked in 5% milk for 1 hour and then probed with antibody against TCTN1(1:200; ab105381; Abcam), or actin (1:4000; M20010; Abmart) as a loading control. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized on G: Box Chemi XR5 (Syngene).

Formalin-fixed, paraffin-embedded tissues were used to construct an tissue microarray (TMA) as described previously [19 (link),20 (link)] (Shanghai Biochip Company). Briefly, after verification with hematoxylin and eosin (H&E) staining, 1.5 mm core punch sample was taken from each specimen and cut as 4-μm-thick sections, which were then deparaffinized. Endogenous HRP activity was blocked with 3% H₂O₂, and antigen retrieval was achieved by boiling in sodium citrate buffer (pH 6.0). After blocking in 10% normal goat serum, immunostaining was performed using a rabbit anti-TCTN1 antibody (ab105381; Abcam) at 1: 50 dilution. Finally, the visualization signal was developed with 3,3’-diaminobenzidine (DAB), and the slides were then counterstained in hematoxylin. As negative controls, the sections were incubated with normal mouse serum instead of the primary antibody. The scores of immunohistochemical staining were evaluated by two independent pathologists in a blinded manner as described previously [21 (link)-23 (link)]. Briefly, the expression of TCTN1 was scored by estimating the proportion of tumor cells with positive staining. High TCTN1 expression was defined as >10% positive staining, while low expression was defined as a proportion of < = 10% positive staining, as described in previous studies [22 (link),24 (link)-31 (link)].