Flow cytometric analysis of Jurkat cells treated with 20μM LY294002 for 2 hours (left, negative) and Jurkat cells treated with 100nM Calyculin A for 40 minutes (right, positive) labeling Phospho-FOXO1A (T24)+ FOXO3A (T32)+ FOXO4 (T28)+ FOXO6 (T26). Cells were fixed and permeabilized. Then stained with the primary antibody (HA722499, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Immunocytochemistry analysis of Jurkat cells treated with 20μM LY294002 for 2 hours (upper, negative) and Jurkat cells treated with 100nM Calyculin A for 40 minutes (lower, positive) labeling Phospho-FOXO1A (T24)+ FOXO3A (T32)+ FOXO4 (T28)+ FOXO6 (T26) with Rabbit anti-Phospho-FOXO1A (T24)+ FOXO3A (T32)+ FOXO4 (T28)+ FOXO6 (T26) antibody (HA722499) at 1/1,000 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-FOXO1A (T24)+ FOXO3A (T32)+ FOXO4 (T28)+ FOXO6 (T26) antibody (HA722499) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.