All lanes : Anti-Pellino 1 antibody [EPR19302] (ab199336) at 1/5000 dilution

Lane 1 : Human PELI1 full length recombinant protein
Lane 2 : Human PELI2 full length recombinant protein
Lane 3 : Human PELI3 full length recombinant protein

Lysates/proteins at 0.01 µg per lane.

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Human PELI 1 full length recombinant protein contain aa1-418 with a His-Tag®. Human PELI 2 full length recombinant protein contain aa1-420 with a His-Tag®. Human PELI3 full length recombinant protein contain aa1-469 with a His-Tag®. All three recombinant human fragment proteins were made in-house.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Pellino 1 with ab199336 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19302] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab199336 at 1/100 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Lane 1 (input): Mouse brain lysate, 10μg
Lane 2 (+): Mouse brain lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of  ab199336 in Mouse brain lysate

Ab199336 immunoprecipitating Pellino in Mouse brain lysate. For western blotting, primary antibody used was ab199336 at 1:500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-Pellino 1 antibody [EPR19302] (ab199336) at 1/1000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Pellino 1 antibody [EPR19302] (ab199336) at 1/1000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

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


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Pellino 1 antibody [EPR19302] (ab199336) at 1/1000 dilution

Lane 1 : Ramos (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : Untreated RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) treated with 1 µg/ml LPS for 6 hours whole cell lysate
Lane 5 : Untreated PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) treated with 1 µg/ml LPS for 6 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: 46 kDa
Observed band size: 46 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression of Pellino 1 can be induced by LPS. (PMID: 21204785).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (Human Burkitt's lymphoma cell line) cells labeling Pellino 1 with ab199336 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining on Ramos cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [EPR19302] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab199336 at 1/100 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Pellino 1 was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab199336 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199336 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: Ramos whole cell lysate 10µg (Input).
Lane 2: ab199336 IP in Ramos whole cell lysate.
Lane 3: Rabbit IgG,monoclonal [EPR19302] - Isotype Control (ab172730) instead of ab199336 in Ramos whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: (A): 30 seconds; (B): 3 minutes.