2.6 Co-immunoprecipitation assays
HEK293T and HaCaT cells (0.5 × 106 cells per well)
were scattered in six-well plates, and treated with the indicated
concentrations of ISX9 or DMSO for 24 h. The pooled of 100 µg
protein-lysates were accrued by using 500 μL RIPA initially (phosphatase
inhibitor cocktails and protease inhibitor freshly added), after that
centrifugation is executed (12,000 rpm for 15 min at 4 °C). After
quantification, the lysates were established with respective antibodies
at 4 °C overnight. By using anti-Flag agarose bead (Bimake), we then
enacted an immunoprecipitation. The immunocomplexes were washed five
times with RIPA buffer and boiled in the SDS-loading buffer. They were
then resolute by 10% SDS-PAGE and relocated to PVDF membranes, stalked
by immunoblotting with the stipulated antibodies.