Anti-GSDMD antibody [EPR19828]

• IP

Supplier Data

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 (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) 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

false

• WB

Lab

Western blot - Anti-GSDMD antibody [EPR19828] (AB209845)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

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

Lane 1:

J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate at 20 µg

Lane 2:

J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) treated with 100ng/ml lipopolysaccharides (LPS) for 3h, then added 300ng/ml BFA for another 3h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 180s

• WB

Lab

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

false

• WB

Supplier Data

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 53 kDa

Observed band size: 32 kDa,53 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-GSDMD antibody [EPR19828] (AB209845)

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).

We are unsure how to define the 37kDa extra band.

Other bands may be the GSDMD cleavage products, you can find them in PMID : 34890644, PMID : 33208231, PMID : 28679757.

Blocking and dilution buffer : 5% NFDM/TBST.

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 120s

• WB

CiteAb

Western blot - Anti-GSDMD antibody [EPR19828] (AB209845)

Western Blotting using Anti-GSDMD antibody [EPR19828], ab209845. Publication image from Leppla, S. H. et al., 2020, Nat Commun, 32029733. Legend direct from paper.

A secreted factor of B. cereus activates the inflammasome independently of HBL.a Immunoblot analysis of pro-caspase-1 (Casp-1), the active caspase-1 p20 subunit, pro-pyroptotic effector protein gasdermin D (GSDMD), the active GSDMD p30 subunit of WT BMDMs left untreated [Medium alone (Med.)] or LPS-primed and assessed either 3 h or 20 h after stimulation with the supernatant of WT B. cereus (B. cer.) or δHbl B. cereus (δHbl) (top; Supernatant (Sup.)) or infection with B. cer. or δHbl (bottom; m.o.i. of 5). Release of IL-1β b, IL-18 c, and cell death d of BMDMs as treated in a. NS, not significant, ***P < 0.001 and ****P < 0.0001 (student’s unpaired t test b–d). Each symbol represents an independent experiment b–d. Data are representative of three independent experiments (n = 3, a–d; mean and s.e.m. in b–d). Source data are provided as a Source Data file.

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• WB

CiteAb

Western blot - Anti-GSDMD antibody [EPR19828] (AB209845)

Western Blotting using Anti-GSDMD antibody [EPR19828], ab209845. Publication image from Leppla, S. H. et al., 2020, Nat Commun, 32029733. Legend direct from paper.

The secreted factor of B. cereus activates the NLRP3 inflammasome.a, Immunoblot analysis of caspase-1, gasdermin D, and GAPDH (loading control) in WT or mutant BMDMs left untreated or LPS-primed and assessed 20 h after stimulation with δHbl supernatant, or after infection with δHbl (m.o.i. of 5). b Release of IL-1β, IL-18, and TNF and death of BMDMs after treatment as in a. c Brightfield microscopy analysis of BMDMs 8 h after stimulation as in a. d Immunofluorescent analysis of ASC speck formation (red) in BMDMs after treatment as in a. Quantification of the prevalence of ASC specks is shown on the right as a percentage of total cells (DAPI). At least 200 cells from each genotype were assessed. Scale bar, 25 µm c, d. Arrowheads indicate pyroptotic cells c or ASC specks d. NS, not significant, ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test b or student’s unpaired t test d). Each symbol represents an independent experiment b, d. Data are representative of three independent experiments (n = 3, a–d; mean and s.e.m. in b, d). Source data are provided as a Source Data file.

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