Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal(AB215191)

ab215191 at 1/30 immunoprecipitating DFNA5/GSDME in EMT6 (mouse mammary carcinoma cell line) whole cell lysate.

Lane 1 (input): EMT6 whole cell lysate (10μg)

Lane 2 (+): ab215191 + EMT6 whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab215191 in EMT6 whole cell lysate

For western blotting, ab215191 at 1/500. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

EMT6 cells were kindly provided by our collaborator Dr. Feng Shao, NIBS

All lanes:

Immunoprecipitation - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (ab215191)

Predicted band size: 54 kDa

Exposure time: 55s

Exposure times:

Lane 1: 15s

Lane 2: 3 min

The band around 35kDa is GSDME-NT which might be cleaved by activated caspase‐3 (PMID: 33589596, PMID: 31347748,PMID: 35623158).

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (ab215191) at 1/1000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 55 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (ab215191) at 0.7 µg/mL

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse thymus tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG) at 1/2000 dilution

Predicted band size: 54 kDa

Observed band size: 55 kDa

Exposure time: 3min

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized SH-SY5Y (human neuroblastoma cell line from bone marrow) cell line labeling DFNA5/GSDME with ab215191 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Different batches of ab215191 were tested on SH-SY5Y (Human neuroblastoma epithelial cell) lysate at 0.6 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 55 kDa.

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (ab215191)

Predicted band size: 54 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

The cell lysates were kindly provided by our collaborator, Dr. Feng Shao, NIBS.

All lanes:

Western blot - Anti-DFNA5/GSDME antibody [EPR19859] - N-terminal (ab215191) at 1/5000 dilution

Lane 1:

SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate at 10 µg

Lane 2:

DFNA5/GSDME knockout SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 54 kDa

Observed band size: 55 kDa

Exposure time: 15s