All lanes : Anti-TLE 1 antibody [EPR9386(2)] (ab183742) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : TLE1 knockout HEK-293T cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 83 kDa

Lanes 1-3: Merged signal (red and green). Green - ab183742 observed at 83 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab183742 Anti-TLE 1 antibody [EPR9386(2)] was shown to specifically react with TLE 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265059 (knockout cell lysate ab257240) was used. Wild-type and TLE 1 knockout samples were subjected to SDS-PAGE. ab183742 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 10000 dilution for 1 hour at room temperature before imaging.

 

ab183742 staining TLE1 in wild-type MCF7 cells (top panel) and TLE1 knockout MCF7 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab183742 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 µg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 µg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).

Immunohistochemical analysis of Human schwannoma, staining TLE 1 with ab183742 at 1/250 dilution. Detected using HRP Polymer for Rabbit IgG and counter-stained using hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-TLE 1 antibody [EPR9386(2)] (ab183742) at 1/1000 dilution

Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : TLE1 knockout MCF7 cell lysate
Lane 3 : SH-SY5Y cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 83 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab183742 observed at 83 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab183742 was shown to react with TLE 1 in western blot. The band observed in the knockout lysate lane below 83 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab183742 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 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 h at room temperature before imaging.

All lanes : Anti-TLE 1 antibody [EPR9386(2)] (ab183742) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TLE1 knockout HeLa cell lysate
Lane 3 : 293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 83 kDa

Lanes 1-3: Merged signal (red and green). Green - ab183742 observed at 83 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab183742 Anti-TLE 1 antibody [EPR9386(2)] was shown to specifically react with TLE 1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264901 (knockout cell lysate ab257241) was used. Wild-type and TLE 1 knockout samples were subjected to SDS-PAGE. ab183742 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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 10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-TLE 1 antibody [EPR9386(2)] (ab183742) at 1/1000 dilution

Lane 1 : SH-SY5Y cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HeLa 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: 83 kDa
Observed band size: 83 kDa

Immunofluorescence analysis of paraformaldehyde-fixed HepG2 cells, staining TLE 1 (green) with ab183742 at 1/100 dilution. Alexa Fluor®488-conjugated goat anti rabbit IgG was used as a secondary antibody at 1/200 dilution. Nuclei were counterstained with DAPI (blue).

Immunohistochemical analysis of Human synovial sarcoma, staining TLE 1 with ab183742 at 1/250 dilution. Detected using HRP Polymer for Rabbit IgG and counter-stained using hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.