*4.7. Western Blotting*

Our previously published western blotting assay procedure was used to evaluate endogenous *MAP1B* expression and the *MAP1B*-knockdown efficiency in RTCC1 and J82 cell lines using primary antibodies against *MAP1B* (1:500, clone AA6; Millipore, Beverly, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GADPH) (6C5, 1:10,000; Millipore, Beverly, MA, USA). Cell lysates with 25 μg of protein were separated using a 4% to 12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA, USA), then transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK) for the immobilization of proteins. Membranes were incubated with tris-buffered saline containing Tween 20 (TBST) buffer and 5% skimmed milk at room temperature for one hour for blocking, followed by exposure to primary antibodies at 4 ◦C overnight against *MAP1B* (1:500, clone AA6; Millipore, Beverly, MA, USA) using GADPH as a loading control (6C5, 1:10,000; Millipore, Beverly, MA, USA). Membranes were incubated with the secondary antibody at room temperature for 1.5 h, and proteins were detected using a chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK).
