Human CXCL10 (IP10) knockout A549 cell lysate

• WB

Lab

Western blot - Human CXCL10 (IP10) knockout A549 cell lysate (AB256888)

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate (30 ug)
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate (30 ug)
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate (30 ug)
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100ng/ml, 32h) and TNF-alpha (ab259410) (10ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate (30 ug)
Lane 5 : THP-1 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate (30 ug)
Lane 6 : THP-1 IFN-y (ab259377) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate (30 ug)

Lanes 1 - 6 : Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-IP10 antibody [EPR20764] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr20764-ab214668'>ab214668</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 2:

Wild-type A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100 ng/ml, 32 h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10 ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 2:

Western blot - Human CXCL10 (IP10) knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-cxcl10-ip10-knockout-thp-1-cell-line-ab277860'>ab277860</a>)

Lane 3:

IP10 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 4:

IP10 knockout A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100ng/ml, 32h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 5:

THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 6:

THP-1 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Predicted band size: 10 kDa

false

• WB

Supplier Data

Western blot - Human CXCL10 (IP10) knockout A549 cell lysate (AB256888)

Lane 1 : Untreated Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate, 40 ug
Lane 2 : Wild-type A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate, 40 ug
Lane 3 : Untreated IP10 knockout A549 whole cell lysate, 40 ug
Lane 4 : IP10 knockout A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate, 40 ug
Lane 5 : Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate, 20 ug
Lane 6 : THP-1 treated with 200ng/ml IFN-y (ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours, whole cell lysate, 20 ug

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

Lanes 1-6 : Merged signal (red and green). Green - ab283681 observed at 11 kDa. Red-loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) was observed at 36 kDa.

ab283681 Anti-TNF Receptor I antibody [EPR24674-12] was shown to specifically react with IP10 in treated wild-type A549 cells. Loss of signal was observed when IP10 knockout cell lines ab266971 (knockout cell lysate ab256888) were used. Wild-type and IP10 knockout samples were subjected to SDS-PAGE. ab283681 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight 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.

All lanes:

Western blot - Anti-IP10 antibody [EPR24674-12] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr24674-12-ab283681'>ab283681</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate at 40 µg

Lane 2:

Wild-type A549 treated with 100 ng/ml IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for the last 6 hours, whole cell lysate at 40 µg

Lane 3:

Untreated IP10 knockout A549 whole cell lysate at 40 µg

Lane 4:

IP10 knockout A549 treated with 100 ng/ml IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for the last 6 hours, whole cell lysate at 40 µg

Lane 5:

Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 6:

THP-1 treated with 200ng/ml IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 10 kDa

Observed band size: 11 kDa

false

• Sanger seq

Unknown

Sanger Sequencing - Human CXCL10 (IP10) knockout A549 cell lysate (AB256888)

Allele-2 : 1 bp insertion in exon2

• Sanger seq

Unknown

Sanger Sequencing - Human CXCL10 (IP10) knockout A549 cell lysate (AB256888)

Allele-1 : 4 bp deletion in exon2