Western blot - Anti-GSDMD antibody [EPR19828] (ab209845)

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: ab209845 - 120 seconds, ab219800 - 20 seconds

Compared with ab209845, ab219800 has a higher affinity in positive samples.

The MW observed is consistent with the literature: PMID 26375003.

The 43 kDa band is likely to be a GSDMD cleavage product by caspase-3 at GSDMD Asp88 (PMID: 30902848, PMID: 28392147, PMID: 30135078).

All lanes:

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate, Batch 1 at 20 µg

Lane 2:

Mouse liver lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 20 µg

Lane 4:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate, Batch 2 at 20 µg

Secondary

All lanes:

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

Predicted band size: 53 kDa

Observed band size: 53 kDa

Immunoprecipitation - Anti-GSDMD antibody [EPR19828] (ab209845)

Gasdermin-D was immunoprecipitated from 0.35 mg of RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus)-derived cells with ectopic expression of ASC were primed with 1μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 μM nigericin treatment under serum starved conditions for 2 h, whole cell lysate with ab209845 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab209845 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

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

Exposure time: 3 minutes.

As a response to LPS stimulation and nigericin treatment, Gasdermin-D is cleaved and Gasdermin-D N-terminal form is detected at 32kDa. Details of RAW 264.7-derived cells with ectopic expression of ASC are described in the literature: PMID 26611636.

The cells were kindly provided by our collaborator Dr. Jiahuai Han, Xiamen University.

Note: The antibody has better affinity to full length Gasdermin-D.

All lanes:

Immunoprecipitation - Anti-GSDMD antibody [EPR19828] (ab209845) at 1/1000 dilution

Lane 1:

RAW 264.7-derived cells with ectopic expression of ASC were primed with 1μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 μM nigericin treatment under serum starved conditions for 2 h at 10 µg

Lane 2:

ab209845 IP in RAW 264.7-derived cells with ectopic expression of ASC were primed with 1μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 μM nigericin treatment under serum starved conditions for 2 h whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (ab172730) instead of ab209845 in RAW 264.7-derived cells with ectopic expression of ASC were primed with 1μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 μM nigericin treatment under serum starved conditions for 2 h whole cell lysate

Predicted band size: 53 kDa

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845)

Blocking/Dilution buffer: 5% NFDM/TBST.

As a response to LPS stimulation and nigericin treatment, Gasdermin-D is cleaved and Gasdermin-D N-terminal form is detected at 32kDa. Details of RAW264.7-derived cells with ectopic expression of ASC are described in the literature: PMID 26611636

The MW observed is consistent with the literature: PMID 26375003.

The cells were kindly provided by our collaborator Dr. Jiahuai Han, Xiamen University.

All lanes:

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845) at 1/1000 dilution

Lane 1:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus)-derived cell line with ectopic expression of ASC (Apoptosis-associated speck-like protein), whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus)-derived cell line with ectopic expression of ASC primed with 1 μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 μM nigericin treatment under serum starved conditions for 2 h, whole cell lysate at 20 µg

Lane 3:

Gasdermin-D knockout RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus)-derived cell line with ectopic expression of ASC whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 53 kDa

Observed band size: 32 kDa, 53 kDa

Exposure time: 1min

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845)

Diluting/Blocking buffer and concentration 5% NFDM/TBST

All lanes:

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845) at 1/1000 dilution

All lanes:

J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) 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: 53 kDa

Observed band size: 53 kDa

Exposure time: 3min

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845)

Different batches of ab209845 were tested on:

1: RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) -derived cell line with ectopic expression of ASC primed with 1 μg/mL lipopolysaccharides (LPS) for 4 h followed by 10 uM nigericin treatment under serum starved conditions for 2 h, whole cell lysate at 5.2 μg/ml.

2: RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) -derived cell line with ectopic expression of ASC (Apoptosis-associated speck-like protein), whole cell lysate at 5.2 μg/ml.

15 μg of lysate was loaded in each lane. Bands observed at 32,53 kDa.

All lanes:

Western blot - Anti-GSDMD antibody [EPR19828] (ab209845)

Predicted band size: 53 kDa