Immunocytochemistry analysis of 293T cells labeling HA tag with Rabbit anti-HA tag antibody (HA750766) at 1/1,000 dilution. 293T cells, transfected with HA-tagged empty control, Nanos homolog 3 (N-terminal) or CLK4 (C-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-HA tag antibody (HA750766) 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.

HA tag was immunoprecipitated from 0.2 mg 293T transfected with HA-tagged Nanos homolog 3 (N-terminal) cell lysate with HA750766 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using M1008-1 at 1/20,000 dilution. Anti-Mouse IgG for IP Nano-secondary antibody (NBI02H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: 293T transfected with HA-tagged Nanos homolog 3 (N-terminal) cell lysate (input) Lane 2: HA750766 IP in 293T transfected with HA-tagged Nanos homolog 3 (N-terminal) cell lysate Lane 3: Mouse IgG instead of HA750766 in 293T transfected with HA-tagged Nanos homolog 3 (N-terminal) cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 35 seconds; ECL: K1801