Human HNRNPD knockout U-2 OS cell lysate

• WB

Lab

Western blot - Human HNRNPD knockout U-2 OS cell lysate (AB273814)

Lane 1 : Wild-type U-2 OS cell lysate 20 μg
Lane 2 : HNRNPD knockout U-2 OS cell lysate 20 μg
False colour image of Western blot : Anti-hnRNP D/AUF1 antibody [EPR24001-12] staining at 1/1000 dilution, shown in green; loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) staining at 1/20000 dilution, shown in red. In Western blot, ab259895 was shown to bind specifically to hnRNP D/AUF1. A band was observed at 40 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in HNRNPD knockout cell line ab273860 (knockout cell lysate ab273814). To generate this image, wild-type and HNRNPD knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (<a href='/en-us/products/primary-antibodies/hnrnp-d-auf1-antibody-epr24001-12-ab259895'>ab259895</a>) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

HNRNPD knockout U-2 OS cell lysate at 20 µg

Lane 2:

Western blot - Human HNRNPD knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-hnrnpd-knockout-u-2-os-cell-line-ab273860'>ab273860</a>)

Predicted band size: 38 kDa

Observed band size: 40 kDa

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• WB

Lab

Western blot - Human HNRNPD knockout U-2 OS cell lysate (AB273814)

Lane 1 : Wild-type U-2 OS cell lysate 20 ug

Lane 2 : HNRNPD knockout U-2 OS cell lysate 20 ug

Lane 3 : Jurkat cell lysate 20 ug

Lane 4 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate 20 ug

Lanes 1 - 4 : Merged signal (red and green). Green - ab50692 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab50692 was shown to react with hnRNP D/AUF1 in wild-type U-2 OS cells in western blot with loss of signal observed in HNRNPD knockout cell line ab273860 (knockout cell lysate ab273814). Wild-type and HNRNPD knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab50692 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at 1.25 μg/ml 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.

All lanes:

Anti-hnRNP D/AUF1 antibody (<a href='/en-us/products/unavailable/hnrnp-dauf1-antibody-ab50692'>ab50692</a>) at 1.25 µg/mL

Lane 1:

Wild-type U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

Lane 2:

HNRNPD knockout U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human HNRNPD knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-hnrnpd-knockout-u-2-os-cell-line-ab273860'>ab273860</a>)

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

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• NGS

Supplier Data

Next Generation Sequencing - Human HNRNPD knockout U-2 OS cell lysate (AB273814)

Knockout achieved by CRISPR/Cas9; X = 5 bp deletion; Frameshift : 100%