Cell death / Inflammation / Western blot

A band on a blot.
A question worth exploring.

Antibody testing and paper notes, connecting experimental observations with published evidence.

PAPER NOTE · 0101 / 22

After GSDMD knockout, caspase-1 is still activated and IL-1β still matures, yet the cells no longer undergo pyroptosis

Introduction: Why do cells swell and burst after an inflammasome activates caspase-1? Before 2015, this question had no answer: none of the known caspase-1 substrates at the time could explain pyroptosis. The authors knocked out the 9 proteins identified by their screen one by one. Only 1 knockout prevented cell death and IL-1β release: GSDMD.

Read paper note

Cell Research · 2015

FROM THE BENCH

Antibody reviews15

Images, conditions and observations from antibody testing.

How to read these reviews

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PyroptosisTHP-1 · 1:3000

Caspase-1

Proteintech / 22915-1-AP

The early blots were clear. This time, p10 appears with a longer exposure, while other cleavage bands show little difference.

Longer exposure helpsRead review ↗

PyroptosisTHP-1 · 1:3000

Caspase-1 p20

ABclonal / A23429

After about three years, p20 is still easy to detect; the inhibitor groups also show an interesting intermediate-sized band.

Cleavage changes visibleRead review ↗

PyroptosisTHP-1 · 1:3000

GSDMD NT

ABclonal / A24059

The p30 NT band is prominent, and its reduction after inhibitor treatment is easy to see.

Cleavage changes visibleRead review ↗

PyroptosisTHP-1 · 1:3000

GSDME

ABclonal / A28230

The full-length band drops markedly while p35 NT is clear, making the processing pattern easy to see.

Cleavage changes visibleRead review ↗

PyroptosisTHP-1 · 1:3000

GSDME NT

ABclonal / A26197

I have had good results with both catalog versions; this test shows clear changes at p35 and approximately 13 kDa.

Cleavage changes visibleRead review ↗

ApoptosisTHP-1 · 1:3000

cIAP1

ABclonal / A19688

After more than a year of use, the background remains clean, with cIAP1 loss visible in several cell-death models.

Clear trendRead review ↗

ApoptosisTHP-1 · 1:3000

XIAP

ABclonal / A20846

The background is fairly clean; full-length XIAP decreases while an approximately 30 kDa band becomes stronger.

Clear trendRead review ↗

ApoptosisTHP-1 · 1:3000

Caspase-3

Beyotime / AF5132

Target bands and changes are visible despite numerous extra bands; p17 is less prominent at 120 min than at 60 min.

Extra bandsRead review ↗

ApoptosisTHP-1 · 1:3000

Cleaved caspase-3 p12

ABclonal / A28349

p12 appears at the expected position, increases clearly after stimulation and decreases or disappears with inhibitors.

Cleavage changes visibleRead review ↗

ApoptosisTHP-1 · 1:3000

Caspase-7

Proteintech / 27155-1-AP

Recent runs have more extra bands, but full-length loss and changes in the smaller bands are still visible.

Additional bands, visible trendRead review ↗

ApoptosisTHP-1 · 1:3000

Caspase-9

ABclonal / A18676

Full-length protein and p10 can be detected together; the faint p10 signal is clearer with a longer exposure.

p10 needs longer exposureRead review ↗

PyroptosisTHP-1 · 1:3000

GSDMD

CST · Cell Signaling Technology / #39754

A favorite after about three years of use, with clear p30 and p43 processing patterns.

Cleavage changes visibleRead review ↗

ApoptosisTHP-1 · 1:3000

BAK: Two-antibody comparison

Beyotime / ABclonal / AF1591 / A23523

Both antibodies give clear target bands and clean backgrounds; total BAK does not noticeably increase within 2 h.

Clean backgroundRead review ↗

ApoptosisTHP-1 · 1:3000

BCL2

Beyotime / AG1225

After about three years of use, target bands remain clear and the background clean; BCL2 is fairly stable in this experiment.

Clean backgroundRead review ↗

ApoptosisTHP-1 · 1:3000

PUMA

Beyotime / AF1204

After about three years of use, the background remains clean; PUMA decreases over this stimulation time course.

Clean backgroundRead review ↗

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A band on a blot.
A question worth exploring.

After GSDMD knockout, caspase-1 is still activated and IL-1β still matures, yet the cells no longer undergo pyroptosis

After testing 14 gene-knockout mouse strains one by one, none supported the popular model of "mitochondrial apoptosis activating NLRP3"

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Mitochondrial apoptosis, considered "immunologically silent," can also activate IL-1β: BAX/BAK engages two pro-inflammatory pathways simultaneously through caspase-3/7

The caspase-1 p20 band on a Western blot is actually an inactive product: how self-cleavage terminates inflammasome activity

Gasdermin pores are not limited to the plasma membrane: the N-terminal fragments of GSDME and GSDMD can also permeabilize mitochondria

Inflammasomes remain functional in the absence of GSDMD: caspase-1 cleaves Bid to initiate apoptosis

The only pair among 28 cytokine combinations that kills macrophages: how TNF-α + IFN-γ triggers PANoptosis

A cleavage site strictly conserved through evolution, yet mice remain healthy after it is mutated: RIPK3 cleavage does not govern necroptosis

Membrane rupture at the end of apoptosis is also programmed: caspase-3 cleaves DFNA5 to execute secondary necrosis

IFN-γ alone does not kill macrophages, but TLR ligands given after priming do: how caspase-8 and mitochondrial apoptosis connect infection to cell death

A proteomics screen reveals that the caspase-1 inflammasome directly cleaves and activates caspase-7

The inflammation of necroptosis is not "leaked out": active MLKL activates NLRP3 through potassium efflux before cell rupture

Bidirectional crosstalk between pyroptosis and apoptosis: without GSDMD, caspase-1 triggers apoptosis, while apoptotic caspases inactivate GSDMD in turn

Apoptosis is not immunologically silent: caspase-8 directly cleaves GSDMD to cause cell lysis, while pannexin-1 drives NLRP3 activation

Effector caspases are more than executors: caspase-3/-7 amplify upstream apoptotic signals through feedback

MLKL phosphorylation does not immediately rupture the membrane: intracellular trafficking determines the speed and threshold of necroptosis

An alternative death program after caspase-1 inhibition: caspase-8 switches to cleaving GSDME, permitting IL-1α release through "incomplete pyroptosis"

GSDME expression determines the outcome: endogenous levels sustain IL-1β release without lysing cells, whereas only high expression leads to pyroptosis

Loss of caspase-8 auto-cleavage: weaker apoptosis, enhanced necroptosis, and lymphopenia

Apoptotic and pyroptotic machinery are redundant in XIAP deficiency: only combined deletion blocks cell death and IL-1β release

GSDMD breaches both mitochondrial membranes before the plasma membrane: mitochondrial damage accelerates and amplifies pyroptosis