All lanes : Anti-PBK/SPK antibody [31/PBK] (ab280209) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : PBK knockout HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 4 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution

Predicted band size: 36 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab280209 observed at 36 kDa. Red - loading control ab52901(Rabbit monoclonal [EP1331Y] to beta Tubulin) observed at 52 kDa.

Lanes 1-2: ab280209 Anti-PBK antibody was shown to react with in HEK-293T cells in Western blot. Loss of signal was observed when PBK knockout sample was used. Wild-type and PBK knockout samples were subjected to SDS-PAGE.

ab280209 and Anti-beta Tubulin antibody [EP1331Y] (ab52901) were incubated at 4° overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) (ab216772) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

Blocking/Dilution buffer: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling PBK/PSK with ab280209 at 1/500 (1.946 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Positive staining in germinal center of human tonsil.

The section was incubated with ab280209 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling PBK/SPK with ab280209 at 1/1000 dilution (0.1 µg) (red) compared with a mouse monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti mouse IgG (Alexa Fluor® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-PBK/SPK antibody [31/PBK] (ab280209) at 1/1000 dilution

Lane 1 : Human testis tissue lysate
Lane 2 : Human heart tissue lysate
Lane 3 : Mouse testis tissue lysate
Lane 4 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 36 kDa

Negative control: Human heart, mouse brain (PMID: 27898655).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:23547718).

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PBK/SPK antibody [31/PBK] (ab280209) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 3 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate
Lane 4 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 5 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 7 : Rat testis tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 36 kDa

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:23547718).

Exposure time: lane 1: 37 secs; lane 2-7: 15 secs.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling PBK/PSK with ab280209 at 1/500 (1.946 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Positive staining in mouse testis.

The section was incubated with ab280209 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling PBK/PSK with ab280209 at 1/500 (1.946 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Positive staining in rat testis.

The section was incubated with ab280209 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.