Par 4 specific antibodies

BT-20 and MDA-MB-468 cells were transiently transfected with plasmids encoding PAR-4 WT-eCFP, PAR-4 D131G-eCFP, PAR-4 (132-340)-eCFP and eYFP-PAR-4 (1-131) for 24 h, washed with PBS and fixed with 3.7% (w/v) paraformaldehyde (PFA)/PBS for 20 min. Cells were permeabilized with PBS containing 0,1% Triton-X-100 for 30 min and blocked in 3% bovine serum albumin (BSA) in PBS for 1 h. Endogenous PAR-4 was stained with PAR-4-specific antibodies (Abcam, 1:100) and visualized with secondary Alexa Fluor 555 conjugated antibodies (1:1000). Hoechst 33258 (2 μg/ml, Sigma) was added and coverslips were mounted with Immu-Mount (Thermo Fisher). Image visualisation was performed with the Zeiss LSM 710 confocal microscope using a LDC-apochromat 40×/1.1 water objective. ZEN 2009 (Zeiss) software was used for image editing.

Cells were grown on glass coverslips (18 mm) in 12 well plates, washed with PBS and fixed with 4% paraformaldehyde / PBS for 30 min. Cells were permeabilized with PBS containing 0,1% Triton-X-100 for 30 min and blocked in 3% bovine serum albumin (BSA) in PBS for 1 h. PAR-4 was stained with PAR-4 specific antibodies (Abcam, 1:100) and visualized with secondary Alexa Fluor® 555 conjugated antibodies (1:1000). Hoechst was added and coverslips were mounted with ImmuMount (Thermo Scientific). Images were examined with a Zeiss LSM 710 confocal microscope with a LDC-apochromat 40/1.1 water objective. ZEN 2009 software (Zeiss) was used for image editing.