Western Blot Protein Detection Protocol

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).