Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: HP1 gamma/CBX3 knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HUVEC (umbilicalvein/vascular endothelium) whole cell lysate (20 µg)
Lane 4: HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab202107 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab202107 was shown to specifically react with HP1 gamma/CBX3 when HP1 gamma/CBX3 knockout samples were used. Wild-type and HP1 gamma/CBX3 knockout samples were subjected to SDS-PAGE. Antibodies ab202107 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-HP1 gamma/CBX3 (citrulline R108) antibody [EPR19802-202] (ab202107) at 1/1000 dilution

Lanes 1 & 3 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with a control vector containing GFP tag treated with 10 mM calcium chloride and 10 µM Ionomycin for 2 hours, whole cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with GFP-tagged PADI4 (WT) expression vector, treated with 10 mM calcium chloride and 10 µM Ionomycin for 2 hours, whole cell lysate
Lane 4 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with GFP-tagged PADI2 (WT) expression vector treated with 10 mM calcium chloride and 10 µM Ionomycin for 2 hours, 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

Predicted band size: 21 kDa
Observed band size: 21 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lanes 1-2: 2 seconds; Lanes 3-4: 6 seconds.

 

 

Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293 (human epithelial cell line from embryonic kidney) cells transfected with GFP-tagged PADI4 (WT) treated with 10 mM CaCl₂ and 10 μM Ionomycin labeling HP1 gamma/CBX3 (citrulline R108) with ab202107 at 1/3000 dilution (right panel) compared with a rabbit monoclonal IgG isotype control (ab172730) (left panel). Goat Anti-Rabbit IgG H+L (Alexa Fluor^® 647) (ab150079) was used as the secondary antibody.

Only CaCl₂ and Ionomycin treated, GFP positive cell population result in a positive signal (right panel, Q2).

HP1 gamma/CBX3 (citrulline R108) was immunoprecipitated from 0.35 mg HEK-293 (human epithelial cell line from embryonic kidney) transfected with GFP-tagged PADI4 cell lysate treated with 10 mM CaCl₂ and 10 μM Ionomycin for 2 hours with ab202107 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab202107 at a dilution of 1/2000. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10000 dilution.

Lane 1: HEK-293 transfected with GFP-tagged PADI4 (WT) expression vector whole cell lysate treated with 10 mM CaCl₂ and 10 μM Ionomycin for 2 hours 10 μg (Input).

Lane 2: ab202107 IP in HEK-293 transfected with GFP-tagged PADI4 (WT) expression vector whole cell lysate treated with 10 mM CaCl₂ and 10 μM Ionomycin for 2 hours.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202107 in HEK-293 transfected with GFP-tagged PADI4 (WT) expression vector whole cell lysate treated with 10 mM CaCl₂ and 10 μM Ionomycin for 2 hours.

Exposure time: 10 seconds.