All lanes : Anti-ADK antibody [EPR23166-143] (ab243636) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ADK knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 41 kDa
Observed band size: 41 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab243636 observed at 41 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab243636 was shown to react with Adenosine kinase in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265646 (knockout cell lysate ab257343) was used. Wild-type HeLa and ADK knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab243636 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-3 (Human prostate adenocarcinoma epithelial cell) cells labeling ADK with ab243636 at 1\500 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling ADK with ab243636 at 1/1000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining on rat kidney is observed. The section was incubated with ab243636 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

All lanes : Anti-ADK antibody [EPR23166-143] (ab243636) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 5 : Mouse liver lysate
Lane 6 : Rat liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 41 kDa
Observed band size: 41 kDa

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

The lower band (38 kDa) in liver lysates represents ADK-short isoform.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 29038540, 30944910).

Exposure time: Lanes 1-4: 10 seconds; Lanes 5-6: 26 seconds.

All lanes : Anti-ADK antibody [EPR23166-143] (ab243636) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3 : Wild-type HAP1 whole cell lysate at 40 µg
Lane 4 : ADK knockout HAP1 whole cell lysate at 40 µg
Lane 5 : Hepa1-6 (mouse hepatoma epithelial cell), whole cell lysate at 20 µg
Lane 6 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 41 kDa

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 29038540, 30944910).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling ADK with ab243636 at 1/1000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining on mouse cerebrum is observed. The section was incubated with ab243636 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling ADK with ab243636 at 1/1000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining on human liver is observed. The section was incubated with ab243636 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling ADK with ab243636 at 1/1000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and weak cytoplasmic staining on human tonsil is observed. The section was incubated with ab243636 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.