Before reprobing, the membranes were stripped with 0.4 M NaOH for 10 min at room temperature, blocked and incubated with the corresponding antibody.
The cells were then permeabilised with 0.3% Triton X-100 for 30 minutes at room temperature, blocked with 1% BSA for one hour at room temperature, and probed with the specific antibodies overnight at 4°C.
Briefly, cells were fixed in 4% formaldehyde at room temperature, blocked in 10% donkey serum, stained with primary antibody, and detected with secondary antibody, all for 1 hr at room temperature.
Blots were washed in Tris Buffered Saline (TBS) for 5 min at room temperature, blocked for 1 h in TBS + 0.1% Tween 20 (TBS/T) plus 5% dry milk at room temperature and then washed three times in TBS/T.
Wells were coated with 50 ng of avidin or RGDPEG-avidin overnight at room temperature, blocked with BSA (1% in PBS) and incubated with 10 ng of biotinylated TRAIL for 2 h at room temperature.
Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, blocked in 3% BSA, stained with the indicated antibodies, and examined with a laser scanning confocal microscope (DMI 4000B; Leica, Wetzlar, Germany).
To detect total ERK, the membrane was stripped with stripping buffer (Thermo Scientific) for 15 min at room temperature, blocked and probed again with the rabbit polyclonal antibody against anti-ERK1/2 (Millipore) as described above.
LoVo cells (2.5 × 10) were fixed for 40 minutes with 4% paraformaldehyde in PBS at room temperature, blocked with 5% non-fat dry milk, and permealized with solution containing 1% BSA, 0.5% Triton X-100.
After citrate epitope retrieval of paraffin sections, tissues were permeabilized for 5 min with 0.2% Triton X-100 at room temperature, blocked for 1 h with 5% FCS and incubated overnight with the appropriate antibody dilution.
