Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate

• Sanger seq

Unknown

Sanger Sequencing - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (AB258080)

Allele-1 : 5 bp deletion in exon 1

• Sanger seq

Unknown

Sanger Sequencing - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (AB258080)

Allele-2 : 1 bp insertion in exon 1

• WB

Lab

Western blot - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (AB258080)

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

Performed under reducing conditions.

In Western blot, ab325540 was shown to bind specifically to NPR1. Target of interest was observed at 130 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in NPR1 knockout cell line (lane 2) (lane 2, knockout cell line ab265930 / knockout cell lysate ab258080).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 6 seconds, lane 3 : 180 seconds

All lanes:

Western blot - Anti-NPR-A/Atrial natriuretic peptide receptor 1 antibody [EPR30453-586] (<a href='/en-us/products/primary-antibodies/npr-a-atrial-natriuretic-peptide-receptor-1-antibody-epr30453-586-ab325540'>ab325540</a>) at 1/1000 dilution

Lane 1:

Parental HeLa(human cervical adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

Western blot - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (ab258080) at 40 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 130 kDa,36 kDa

false

• WB

Lab

Western blot - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (AB258080)

Lane 1 : Wild-type HeLa cell lysate 20 μg
Lane 2 : NPR1 knockout HeLa cell lysate 20 μg
Lane 3 : U-251 MG cell lysate 20 μg
Lane 4 : Human Heart tissue lysate 20 μg

Lanes 1 - 4 : Merged signal (red and green). Green - anti-NPR1 antibody observed at 140 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
anti-NPR1 antibody was shown to react with NPR1 in wild-type HeLa cells in Western blot with loss of signal observed in NPR1 knockout cell line ab265930 (NPR1 knockout cell lysate ab258080). Wild-type HeLa and NPR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with anti-NPR1 antibody and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4^°C at a 1 in 500 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-NPR1 antibody at 1/500 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (ab258080) at 20 µg

Lane 3:

U-251 MG cell lysate at 20 µg

Lane 4:

Human Heart tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

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 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

false