All lanes : Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : ANP32B knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Lncap cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa

Lanes 1-4: Merged signal (red and green). Green - ab200836 observed at 29 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab200836 Anti-PHAPI2 / APRIL antibody [EPR14588] was shown to specifically react with PHAPI2 / APRIL in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266818 (knockout cell lysate ab257831) was used. Wild-type and PHAPI2 / APRIL knockout samples were subjected to SDS-PAGE. ab200836 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling PHAPI2 / APRIL with ab200836 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human prostatic hyperplasia tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling PHAPI2 / APRIL with ab200836 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and Ab195889 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/200 dilution (red). Confocal image showing mostly nuclear staining in Raw264.7 cell line.

Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/1000 dilution + PC-3 (Human prostate cancer cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/10000 dilution

Lane 1 : LNCaP (Human prostate cancer cell line) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PHAPI2 / APRIL antibody [EPR14588] (ab200836) at 1/1000 dilution

Lane 1 : PC-3 (Human prostate cancer cell line) whole cell lysate
Lane 2 : PC-3 (Human prostate cancer cell line) whole cell lysate with PHAPI2 / APRIL peptide
Lane 3 : PC-3 (Human prostate cancer cell line) whole cell lysate with PHAPI peptide

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on sequence analysis ab200836 has 78% homology with PHAPI protein. The levels of XR were tested in the accompanying blocking experiment.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling PHAPI2 / APRIL with ab200836 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear and cytoplasmic staining on Jurkat cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
-ve control 1: ab200836 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling PHAPI2 / APRIL with ab200836 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human spleen tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling PHAPI2 / APRIL with ab200836 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Mouse spleen tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cardiac muscle tissue labeling PHAPI2 / APRIL with ab200836 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Rat cardiac muscle tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling PHAPI2 / APRIL with ab200836 at 1/180 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.