For western blot analysis, HEK293T cells were transfected with the indicated construct and harvested after washing in PBS. The cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5% glycerol), supplemented with protease and phosphatase inhibitor cocktails. Homogenates were cleared by centrifugation at 13,200 rpm for 15 minutes at 4 °C. The lysates were subsequently used for either co-immunoprecipitation experiment or western blot analysis. For the western blot analysis, the samples were run onto 6–12% polyacrylamide gel. Blots were blocked in 5% TBS + 0.05% Tween 20 and incubated with anti-DDDD-K (Abcam) or anti-HA (Roche) antibodies. Proteins were visualized using HRP-conjugated secondary antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo) and exposed to ChemiDoc MP (Bio-Rad).
For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700.
For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700.