All lanes : Anti-IP10 antibody [EPR7850] (ab137018) at 1/500 dilution

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

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

Predicted band size: 10 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab137018 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab137018 was shown to react with IP10 in A549 wild-type cells in western blot with loss of signal observed in IP10 knockout cell line ab266969 (IP10 knockout cell lysate ab256886). A549 wild-type and IP10 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab137018 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^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Wild-type A549 control cells or IP-10 knockout A549 cells (ab266969), grown to 40% confluency, were stimulated with Recombinant Human Interferon gamma protein (ab259377) at 100 ng/ml and Recombinant human TNF alpha protein (ab259410) at 10 ng/ml or vehicle control for 16 or 32 hours.

THP-1 cells, grown to 40% confluency, were stimulated with Recombinant Human Interferon gamma protein (ab259377) at 200 ng/ml and LPS at 50 ng/mL or vehicle control for 24 hours.

The concentrations of IP-10 (CXCL10) in cell culture supernatants were measured in duplicate and interpolated from the IP-10 standard curves. IP-10 from vehicle control samples were measured in undiluted supernatants and the treated samples were diluted 200 times. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).

Homozygous: 1 bp insertion in exon2