All lanes : Anti-IRF5 antibody [EPR23312-91] (ab274373) at 1/1000 dilution

Lanes 1 & 5 : THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lanes 2 & 7 : A20 (mouse reticulum sarcoma b lymphocyte) whole cell lysate
Lane 3 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 4 : MOLT-4 (human lymphoblastic leukemia t lymphoblast) whole cell lysate
Lane 6 : Ramos (human burkitts lymphoma b lymphocyte) whole cell lysate
Lane 8 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) 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: 56 kDa
Observed band size: 56 kDa

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

Lysates loaded onto lanes 1 and 2 were made freshly and used in WB immediately to minimize protein degradation. Bands beneath the target band in lanes 5 and 7-8 may be caused by degradation.

Negative control: K-562 and MOLT-4 (PMID: 11303025).

The blot of lanes 5-8 was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (Human monocytic leukemia monocyte) cells labelling IRF5 with ab274373 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic and weak nuclear staining in THP-1 cells is observed.

Tubulin is detected with Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) cells labelling IRF5 with ab274373 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG  isotype control (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). 

A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

IRF5 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab274373 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab274373 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 whole cell lysate 10 ug.

Lane 2: ab274373 IP in THP-1 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab274373 in THP-1 whole cell lysate

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

Exposure time: 15 seconds.

Lysate was made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

All lanes : Anti-IRF5 antibody [EPR23312-91] (ab274373) at 1/1000 dilution

Lane 1 : Mouse spleen tissue lysate
Lane 2 : Mouse thymus tissue lysate
Lane 3 : Mouse lymph node tissue lysate
Lane 4 : Rat thymus tissue lysate
Lane 5 : Rat lymph node tissue lysate
Lane 6 : NR8383 (rat norvegicus lu macrophage (alveolar)) 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: 56 kDa
Observed band size: 56 kDa

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

Bands beneath the target band may be caused by degradation.

The blot was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A20 (Mouse reticulum sarcoma B lymphocyte) cells labelling IRF5 with ab274373 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic and weak nuclear staining in A20 cells is observed.

Tubulin is detected with Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized A20 (Mouse reticulum sarcoma B lymphocyte) cells labelling IRF5 with ab274373 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

IRF5 was immunoprecipitated from 0.35 mg A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate with ab274373 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab274373 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A20 whole cell lysate 10 ug.

Lane 2: ab274373 IP in A20 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab274373 in A20 whole cell lysate.

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

Exposure time: 15 seconds.

Lysate was made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.