Western Blotting
Cells for western blotting were plated on 6-well plates (Corning). 48
hours post-transfection, Hela cells and NIH3T3 cells were rinsed with
ice-cold PBS, collected with cell scrapers and lysed with RIPA buffer
(Thermo Scientific, 89900) with protease inhibitor (Sigma, 11836170001)
for 30 minutes on ice, and were then centrifuged at 14000rpm for 20
minutes. The supernatant was transferred to a new tube to which SDS
loading buffer was added and mixed before being boiled for 10 minutes at
95℃. Western Blotting was then performed as previously described. The
proteins were immunoblotted with GFP antibody (1:1000, Santa Cruz
Biotechnology, sc-9996), FOLR1 antibody (1:1000, Invitrogen, PA5-86666),
GAPDH antibody (Cell Signaling Technology, 5174S), Vangl2 antibody
(1:1000, Proteintech, 21492-1-AP), RhoA antibody (1:500, Santa Cruz
Biotechnology, sc-418) and CIC antibody (Cat#: A301-204A, Bethyl, TX)
overnight at 4℃. On the second day, the membrane was incubated with
HRP-conjugated anti-rabbit antibody or anti-mouse antibody (1:5000, Cell
Signaling Technology, 7074S and 7076S) for 1 hour at room temperature.
Membranes were rinsed with ECL substrates (Thermo Scientific, 34580) and
protein bands were detected using a BioRad Gel Doc XR+ Imaging System.