All lanes : Anti-IL-17A antibody [EPR21776] (ab218013) at 1/1000 dilution

Lane 1 : Untreated EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 2 : EL4 treated with 50 ng/ml Phorbol-12-myristate-13-acetate (PMA) and 500 ng/ml ionomycin (ab120116) for 6 hours, then with 500 ng/ml Brefeldin A for 18 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Observed band size: 14,17 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

Expression of IL-17A can be induced by PMA and Ionomycin treatment (PMID 28382171).

The expression profile is consistent with the literature (PMID 9764847).

All lanes : Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (ab219406) at 1/10000 dilution

Lane 1 : Whole cell lysate from HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours
Lane 2 : Whole cell lysate from HEK-293T cells transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours

Lysates/proteins at 10 µg per lane.

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

Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% BSA/TBST.

Histone H3R8 is citrullinated by PADI4 and CaCl₂ is used as a cofactor according to the literature (PMID: 16567635). Ionomycin is used to improve the modification by PADI4 according to the literature (PMID: 26360112).

All lanes : Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (ab219406) at 1/5000 dilution

Lane 1 : Whole cell lysate from NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 for 2 hours
Lane 2 : Whole cell lysate from NIH/3T3 transfected with PADI4 (WT) then treated with 10mM CaCl2 for 2 hours
Lane 3 : Whole cell lysate from C6 (Rat glial tumor cell line) transfected with empty vector with GFP tag (vector control) then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours
Lane 4 : C6 transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours

Lysates/proteins at 10 µg per lane.

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

Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% BSA/TBST.

Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector (left panel) or PADI4 (WT, right panel), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, labeling Histone H3 (citrulline R8) with ab219406 at 1/500 dilution, followed by Goat anti rabbit IgG (Alexa Fluor^® 647) at 1/2000 dilution.

Positive signal is obtained from HEK-293T cells transfected with WT PADI4 treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours.

Histone H3 (citrulline R8) was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate with ab219406 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab219406 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate 10 µg (Input).
Lane 2: ab219406 IP in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219406 in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/5000 dilution

Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate
Lane 2 : HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% BSA/TBST.

Histone H3R17 is citrullinated by PADI4 and CaCl₂ is used as a cofactor according to the literature (PMID: 16567635). Ionomycin is used to improve the modification by PADI4 according to the literature (PMID: 26360112).

All lanes : Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/5000 dilution

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 for 2 hours, whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with PADI4 (WT) then treated with 10mM CaCl2 for 2 hours, whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) transfected with empty vector with GFP tag (vector control) then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate
Lane 4 : C6 (Rat glial tumor cell line) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% BSA/TBST.

Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector (left panel) or PADI4 (WT, right panel), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, labeling Histone H3 (citrulline R17) with ab219407 at 1/500 dilution, followed by Goat anti rabbit IgG (Alexa Fluor^® 647) at 1/2000 dilution.

Positive signal is obtained from HEK-293T cells transfected with WT PADI4 treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours.

Histone H3 (citrulline R17) was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate with ab219407 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab219407 at 1/1000 dilution.

VeriBlot for IP secondary antibody (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate 10 µg (Input).
Lane 2: ab219407 IP in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219407 in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

ab58668 staining ATF3 in A549 cells treated with ionomycin Ca²⁺ salt (ab120116), by ICC/IF. Increase in ATF3 expression correlates with increased concentration of ionomycin Ca²⁺ salt, as described in literature.
The cells were incubated at 37°C for 2h in media containing different concentrations of ab120116 (ionomycin Ca²⁺ salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab58668 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Sandwich ELISA - IFN gamma Human ELISA Kit (ab46025)

Jurkat were stimulated for 48 hours with 50 ng x mL-1 of PMA (ab120297) and 1 uM Ionomycin (ab120116) and PBMCs were stimulated for 48 hours with 2 % PHA-M (LifeTechnologies). Cell free supernatants were tested, showing results after background signal was subtracted (duplicates +/- SD).