All lanes : Anti-Interferon gamma antibody [EPR23991-53] (ab267369) at 1/1000 dilution

Lane 1 : Untreated NK-92 (human malignant non-hodgkins lymphoma natural killer cell) whole cell lysate
Lane 2 : NK-92 treated with 80nM PMA and 3uM Ionomycin for 5 hours, then with /ml Brefeldin A (BFA) added after 1 hour, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 19 kDa

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

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID:23129404)

Exposure time: 92 seconds

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NK-92 cells labelling Interferon gamma with ab267369 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NK-92 treated with phorbol 12-myristate 13-acetate (80 nM) and lonomycin (3 µM) for 1 hour and then treated with brefeldin A (300 ng/ml) for another 4 hours is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NK-92 (Human malignant non-Hodgkin's lymphoma natural killer cell) treated with phorbol 12-myristate 13-acetate (PMA, 80 nM) and lonomycin (3 µM) for 1h and then co-treated with brefeldin A (300 ng/ml) for another 4h cells labelling Interferon gamma with ab267369 at 1/500 dilution (0.1ug) (Right) / compared with Untreated control cells (Left). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with cell stimulation cocktail (80nM PMA+1.34uM Ionomycin+10.6uM Brefeldin A+2uM Monensin) for 6 hours cells labelling Interferon gamma with ab267369 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were stained with anti-CD4 conjugated to Pacific blue. Fixed with 2% PFA for 10 min followed by intracellularly staining with rabbit IgG or ab267369.

Interferon gamma was immunoprecipitated from 0.35 mg NK-92 treated with 80nM PMA and 3µM Ionomycin for 5 hours, then with 300 ng/ml Brefeldin A (BFA) added after 1 hour, whole cell lysate 100 ug with ab267369 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267369 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NK-92 treated with 80nM PMA and 3µM Ionomycin for 5 hours, then with 300 ng/ml Brefeldin A (BFA) added after 1 hour, whole cell lysate 100 ug

Lane 2: ab267369 IP in NK-92 treated with 80nM PMA and 3µM Ionomycin for 5 hours, then with 300 ng/ml Brefeldin A (BFA) added after 1 hour, whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267369 in NK-92 treated with 80nM PMA and 3µM Ionomycin for 5 hours, then with 300 ng/ml Brefeldin A (BFA) added after 1 hour, whole cell lysate

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

Exposure time: 24 seconds