Human FTH1 knockout HEK-293T cell line

• WB

Lab

Western blot - Human FTH1 knockout HEK-293T cell line (AB266581)

Lanes 1-3 : Merged signal (red and green). Green - ab75973 observed at 21 kDa. Red - loading control ab8245 observed at 36 kDa.

ab75973 Anti-Ferritin heavy chain antibody [EPR3004Y] was shown to specifically react with Ferritin heavy chain in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266581 (knockout cell lysate ab256924) was used. Wild-type and Ferritin knockout samples were subjected to SDS-PAGE. ab75973 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (<a href='/en-us/products/primary-antibodies/ferritin-heavy-chain-antibody-epr3004y-ab75973'>ab75973</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human FTH1 knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-fth1-knockout-hek-293t-cell-lysate-ab256924'>ab256924</a>) at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa,36 kDa

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• WB

Lab

Western blot - Human FTH1 knockout HEK-293T cell line (AB266581)

Western blot : Anti-FTH1 antibody (ab65080) staining at 1 ug/ml, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab65080 was shown to bind specifically to FTH1. A band was observed at 21 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in FTH1 knockout cell line. To generate this image, wild-type and FTH1 knockout HEK-293T 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Ferritin Heavy Chain antibody (<a href='/en-us/products/primary-antibodies/ferritin-heavy-chain-antibody-ab65080'>ab65080</a>) at 1 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

FTH1 knockout HEK-293T cell lysate at 20 µg

Lane 3:

Wild-type MCF7 ab269599 cell lysate at 20 µg

Lane 4:

FTH1 knockout MCF7 ab269493 cell lysate at 20 µg

Lane 5:

SH-SY5Y cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 21 kDa

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• Sanger seq

Unknown

Sanger Sequencing - Human FTH1 knockout HEK-293T cell line (AB266581)

Homozygous : 5 bp deletion in exon 1