All lanes : Anti-HuR / ELAVL1 antibody [EPR17397] (ab200342) at 1/5000 dilution

Lane 1 : Wild-type SW480 cell lysate
Lane 2 : ELAVL1 knockout SW480 cell lysate
Lane 3 : Wild-type HAP1 cell lysate at 40 µg
Lane 4 : ELAVL1 knockout HAP1 cell lysate at 40 µg

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab200342 observed at 36 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab200342 was shown to react with ELAVL1 in wild-type SW480 cells in western blot with loss of signal observed in ELAVL1 knockout sample. Wild-type and ELAVL1 knockout SW480 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab200342 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab200342 staining HuR / ELAVL1 in Jurkat (human acute T cell leukemia) cells by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/23000. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

All lanes : Anti-HuR / ELAVL1 antibody [EPR17397] (ab200342) at 1/5000 dilution

Lane 1 : 293 (Human embryonic kidney) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 4 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate

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: 36 kDa
Observed band size: 36 kDa


Exposure time: 5 seconds

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HuR / ELAVL1 antibody [EPR17397] (ab200342) at 1/1000 dilution

Lane 1 : Human fetal brain 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: 36 kDa
Observed band size: 36 kDa


Exposure time: 5 seconds

Blocking/dilution buffer: 5% NFDM/TBST.

Anti-HuR / ELAVL1 antibody [EPR17397] (ab200342) at 1/1000 dilution + Human fetal heart lysate at 10 µg

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

Predicted band size: 36 kDa
Observed band size: 36 kDa


Exposure time: 1 minute

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HuR / ELAVL1 antibody [EPR17397] (ab200342) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat spleen 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: 36 kDa
Observed band size: 36 kDa


Exposure time: 5 seconds

Blocking/dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling HuR / ELAVL1 with ab200342 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasm staining on Human cervix carcinoma 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).

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

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling HuR / ELAVL1 with ab200342 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasm staining on Mouse 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).

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

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling HuR / ELAVL1 with ab200342 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear and weakly cytoplasm staining on rat cerebral cortex tissue 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).

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

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling HuR / ELAVL1 with ab200342 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.

HuR / ELAVL1 was immunoprecipitated from 1mg of  HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab200342 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab200342 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell lysate 10 µg (Input). Lane 2: ab200342 IP in HeLa whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200342 in HeLa whole cell lysate.

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