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

Predicted band size: 15 kDa
Observed band size: 15 kDa


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).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP only or a GFP-tagged PADI4 expression construct, then treated with 10mM CaCl₂ for 2 hours, labeling Histone H3 (citrulline R8) with ab219406 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing positive staining on HEK-293T cells transfected with a GFP-tagged PADI4 expression construct, then treated with 10mM CaCl₂ for 2 hours.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

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

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% BSA/TBST.

All lanes : Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (ab219406) at 1/1000 dilution

Lane 1 : E12 rat embryo lysate
Lane 2 : E12 mouse embryo lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 15 kDa
Observed band size: 15 kDa


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.

Direct ELISA using ab219406 at 0-1000 ng/ml, followed by Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) at 1/2500 dilution. Antigen concentration: 1000 ng/ml.