EMBO/soda-nel huggingface dataset:
GEMC1 is a critical regulator of multiciliated cell differentiation (2016)
Figure 7

Figure 7. GEMC1 transcriptionally activates the MCC program. A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3). B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3). C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells. D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed. E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3. G. Model for the role of GEMC1 in the differentiation of MCCs. Inhibition of Notch signaling by Mir34/449 is required for MCC differentiation and the ability of GEMC1 and Multicilin to activate transcription(Kyrousi et al, 2015; Liu et al, 2007; Marcet et al, 2011; Stubbs et al, 2012; Tsao et al, 2009). GEMC1 acts early, in conjunction with E2F4/5-DP1 (EDG), to activate genes including Multicilin and FoxJ1, likely through direct interaction with E2F4 at the promoters of FoxJ1 and some other E2F target genes (dotted line) (Fig 5, 6 and 7). The activation of Multicilin leads to further amplification of the transcriptional program through E2Fs (EDM complex)(Ma et al, 2014) and GEMC1 and Multicilin may form heterocomplexes (Fig 6E) with E2F4/5-DP1 (EDM-EDG) (Caillat et al, 2013). This leads to the optimal activation of FOXJ1, MYB, RFX3, and other transcription factors required for the expression of CCDC78, DEUP1, CCNO and other downstream components of the MCC differentiation pathway(Funk et al, 2015; Klos Dehring et al, 2013; Ma et al, 2014; Tan et al, 2013; Wallmeier et al, 2014; Zhao et al, 2013). See discussion for further details.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 1

Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome 18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 2

Figure 2. Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH. (B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel). (C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank’s buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter. (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at 18q (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 3

Figure 3. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector. (B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide. (C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower). (D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 4

Figure 4. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells. (C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating. (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel). (E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue). (F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell. (G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomere DNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay). (H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 5

Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116 p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome 18q subtelomere (panel A) or 13q subtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP-qPCR for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs. (C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. (E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 6

Figure 6. Temporal analysis the p53-dependent γH2AX and transcription response to DNA damage stress. (A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment. (B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere 18q as indicated below. Lower panel shows the No-RT control for 18q subtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs. (E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3 ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere. (F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.
Subtelomeric p53 Binding Prevents Accumulation of DNA Damage at Human Telomeres (2015)
Figure 7

Figure 7. CRISPR-deletion of 18q p53-binding site reveals local changes in transcription and γH2AX formation in response to DNA damage. (A) Schematic showing the deletion of ~70 bp region covering non-canonical p53 binding site and adjacent (TG)n repeat in chromosome 18q. (B) Western blot analysis of CRISPR deleted cell lines 18q WT and 18q Δp53 treated with DMSO (D) or 50 μM etoposide (E) for 24 hr. Blots were probed with pan-p53, phospho-p53, γH2AX, or GAPDH (as loading control). (C-E) RT-qPCR for p21 (C), PARDG6 (D), or 18q subtelomeric RNA (E) in 18q WT or18q Δp53 treated with DMSO (black) or 50 μM etoposide (red) for 24 hr. (F) ChIP-qPCR assay for γH2AX at 18q subtelomere in 18q WT or 18q Δp53 treated as in panels B-E.
Time-series transcriptomics and proteomics reveal alternative modes to decode p53 oscillations (2022)
Figure 1

Figure 1: Quantification of mRNA and protein dynamics under oscillatory and rising p53 levels

(a) Schematic showing the negative feedback loop between p53 and MDM2 and the induction of p53 oscillatory or rising dynamics using irradiation with or without the MDM2 inhibitor Nutlin-3a, respectively. Samples for mRNA-seq and mass-spectrometry were collected at the indicated time points following radiation.

(b-c) Western blot for p53 and select p53 targets post-radiation in (b) oscillatory and (c) rising conditions. Actin is shown as a loading control.

(d-e) Average levels of (d) mRNA quantified by RNAseq (red) or qRT-PCR (grey) or (e) protein quantified by mass spectrometry (light green) or western blot (grey) for select p53 target genes under oscillatory p53.

(f-g) Average levels of (f) mRNA quantified by RNAseq (blue) or qRT-PCR (grey) or (g) protein quantified by mass spectrometry (dark green) or western blot (grey) for select p53 target genes under rising p53.

Data information: In d-g, n=2 biological replicates, error bars represent std dev.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 1

Figure 1. Abro1−/− macrophages show defective activation of the NLRP3 inflammasome.

A, B LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) and ELISA of IL-18 (B) in the culture supernatants.

C CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody.

D ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies.

E Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with nigericin, silica, MSU, MDP, Alum, poly (dA: dT), flagellin or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants.

F Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin or poly (dA: dT).

G Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times.

H Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly (dA:dT). Scale bars, 10 μm.

I Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable.

J LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody.

Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (A-E, G, I) or are representative of three independent experiments (F, H, J). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; two-tailed unpaired t-test (C-E, I) or two-way ANOVA with Bonferroni post-test (A, B, G).

Source data are available online for this figure.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 2

Figure 2. ABRO1 deficiency attenuated NLRP3-dependent responses in vivo.

A, B WT, Abro1−/− and Nlrp3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (A). Flow cytometric analysis of peritoneal cell exudates (B). n = 6 for PBS groups and n = 12 for MSU groups.

C Abro1+/+ and Abro1−/− mice were intraperitoneally injected with MSU (1 mg per mouse). Flow cytometric analysis of caspase-1 activity in peritoneal exudate monocytes and neutrophils. n=12 per group.

D, E Abro1+/+ and Abro1−/− mice were intraperitoneally injected with Alum (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β in the peritoneal lavage fluid (D) and flow cytometric analysis of peritoneal cell exudates (E). n = 6 for PBS groups and n = 12 for Alum groups.

F Survival of Abro1+/+ and Abro1−/− mice subjected to LPS (15 mg/kg). n=20 per group.

G ELISA of serum IL-1β from Abro1+/+ and Abro1−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.

Data information: Data are pooled from two (all PBS groups) or three (C, F, MSU groups of A-B, Alum groups of D-E and LPS groups of G) independent experiments. In (A-E and G), data are shown in box-and-whisker plots: dots indicate individual mice; Box-plot center line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. *P < 0.05, **P < 0.01, ***P < 0.001; log-rank test (F) or two-tailed unpaired t-test (A-E, G).

Source data are available online for this figure.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 3

Figure 3. ABRO1 interacts with NLRP3.

A HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.

B-E BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.

F A schematic representation of NLRP3 wild type and deletion mutants.

G HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.

H A schematic representation of ABRO1 wild type and deletion mutants.

I HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.

Data information: Data are representative of three independent experiments.

Source data are available online for this figure.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 4

Figure 4. Phosphorylation of serine 194 in NLRP3 modulates the interaction with ABRO1.

A HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.

B WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.

C WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.

D HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.

E HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.

F WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.

Data information: Data are representative of three independent experiments.

Source data are available online for this figure.
ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination (2019)
Figure 5

Figure 5. ABRO1 promotes deubiquitination of NLRP3.

A HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.

B HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.

C LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.

D Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.

E HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.

F Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.

G HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.

H LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.

Data information: Data are representative of three independent experiments.

Source data are available online for this figure.