All lanes : Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/20000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
Lane 3 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 91 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling STAT5a + STAT5b with ab194898 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing both cytoplasmic and nuclear staining on K562 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab194898 at 1/250 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

All lanes : Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/2000 dilution

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

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: 91 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/1000 dilution + Human STAT5A full length recombinant protein at 10 µg

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

Predicted band size: 91 kDa

Recombinant human C-terminal Myc/DDK tagged full length STAT5A from a commercial source.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/2000 dilution

Lane 1 : mouse heart lysates
Lane 2 : mouse kidney lysates
Lane 3 : mouse spleen lysates
Lane 4 : rat brain lysates
Lane 5 : rat heart lysates
Lane 6 : rat spleen lysates
Lane 7 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 8 : PC12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 9 : NIH3T3 (Mouse embyro fibroblast cells) whole cell lysates

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: 91 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of human tonsil is observed.

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 STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of mouse spleen is observed.

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

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image: negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of rat spleen is observed.

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 K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling STAT5a + STAT5b with ab194898 at 1/80 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and a 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.

STAT5a and STAT5b were immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab194898 at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

STAT5a + STAT5b were co-immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab32043 (Rabbit monoclonal [E289] to STAT5a) at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

STAT5a + STAT5b were co-immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab178941 (Rabbit monoclonal [EPR16671] to STAT5b) at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.