Co-Immunoprecipitation and Western Blot assays
Cells were harvested from 10 cm dishes 24 h after transfection and lysed
with 500 μl lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH=7.5), 1%
Triton X-100, 1% sodium deoxycholate, 1× PMSF, and sodium fluoride).
Suspended cell lysates (400 μl) was taken to incubate with anti-Flag M1
agarose affinity gel (Sigma-Aldrich, A4596-5ml) for 4 h or overnight at
4 ℃. The immunoprecipitates were washed five times with 1 ml lysis
buffer for 5 min each and then boiled at 98 ℃ for 10 min in 65 μl 2×
sample loading buffer. The rest cell lysates (100 μl) were diluted with
5× sample loading buffer and boiled at 98 ℃ for 10 min, followed by
western blot analysis.
The samples were subjected to SDS-PAGE and then transferred to 0.45 μm
Polyvinylidene fluoride (PVDF) membrane (Merck millipore, IPVH00010).
PVDF membranes were blocked in 5% non-fat milk prepared with
Tris-buffered salined with Tween 20 (TBST) for 1 h, and incubated with
corresponding primary antibody, second HRP-linked IgG at room
temperature in 5% non-fat milk for 2 h each successively. After each
incubation, PVDF membranes were washed five times with TBST for 5 min
each. Target proteins were detected with ECL reagent (Tanon,
XSK-180-5001) and photographed by chemiluminescence imager (Tanon,
5200).