Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Ramos (Human Burkitt's lymphoma B lymphocyte) cells labelling Pellino 1+ Pellino 2 with ab254297 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control 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.

All lanes : Anti-Pellino 1 + Pellino 2 antibody [EPR23275-22] (ab254297) at 1/1000 dilution

Lane 1 : Daudi (human burkitts lymphoma lymphoblast) whole cell lysate
Lane 2 : Untreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : RAW 264.7 treated with 100 ng/ml LPS for 7 hours whole cell lysate
Lane 4 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Lanes 3-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 46 kDa
Observed band size: 46 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: 25295537, 21204785, 28009353)

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes.

Pellino 1+ Pellino 2 was immunoprecipitated from 0.35 mg Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate with ab254297 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using 254297 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate 10 ug

Lane 2: 254297 IP in Daudi whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab254297 in Daudi whole cell lysate

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

Exposure time: 110 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Daudi (Human Burkitt's lymphoma lymphoblast) cells labelling Pellino 1+ Pellino 2 with ab254297 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control 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.

All lanes : Anti-Pellino 1 + Pellino 2 antibody [EPR23275-22] (ab254297) at 1/5000 dilution

Lane 1 : His-tagged human PELI1 full length recombinant protein 20 ng
Lane 2 : His-tagged human PELI2 full length recombinant protein 20 ng
Lane 3 : His-tagged human PELI3 full length recombinant protein 20 ng

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

Predicted band size: 46 kDa
Observed band size: 46 kDa

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

Samples loaded in lanes 2 and 3 are extracts of E.coil expressing either PELI2 or PELI3. 

Exposure time: 10 seconds.

All lanes : Anti-Pellino 1 + Pellino 2 antibody [EPR23275-22] (ab254297) at 1/1000 dilution

Lane 1 : Ramos (human burkitts lymphoma b lymphocyte) whole cell lysate
Lane 2 : Raji (human burkitts lymphoma b lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 46 kDa
Observed band size: 46 kDa

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

Low expression control: Raji (PMID: 25295537).

The expression profile and molecular weight observed is consistent with what has been described in the literature (PMID: 25295537, 21204785, 28009353).

Exposure time: 3 minutes.