Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) Survival after intraperitoneal injection of LPS in WT and Irak1 −/− mice. Log-Rank Mantel-Cox Test. (B) Transcriptional response to single TLR ligands Poly(I:C), P3C and Kdo-2 Lipid A in BMDMs 0, 1, 4 and 8 h after stimulation. Median from two experiments. Similarity Matrix – Pearson’s Correlation Coefficient. mRNA levels were assayed by Fluidigm microfluidic qPCR. See Table S1. (C) Survival after infection with Yersinia pseudotuberculosis in WT and Irak1 −/− littermate mice. Log-Rank Mantel-Cox Test. (D) Survival correlation with Day 3 serum cytokine levels in mice infected with Yersinia pseudotuberculosis . Pearson’s Correlation Coefficient (E) Cytokines from broncho-alveolar lavages of WT and Irak1 −/− mice 16 h after intranasal injection of Pseudomonas aeruginosa . (Panel E) Data are represented as mean ± SD. Unpaired t test with Welch’s correction. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Injection, Infection

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) Images of IRAK1 in iBMDMs 0, and 2h after infection with GFP expressing Salmonella enterica serovar Typhimurium (MOI10). Scale Bar: 10 μ . (B) Quantification of IRAK1 clusters in iBMDMs on multi-PRR stimulation resulting from treatment with heat-killed Gram positive and Gram-negative bacteria for 0, 2 and 4h. Data are represented as median ± MAD (C) Images of IRAK1 in iBMDMs 0 and 2 h after co-stimulation with P3C and Kdo-2 Lipid A (50 nM each). Scale Bar: 10 μ . (D) Quantification of IRAK1 clustering in iBMDMs 0, 30, 90, and 180 min after pairwise stimulation with TLR ligands – Kdo-2 Lipid A, P3C, Poly(I:C) and R848. Data are represented as mean (n = 3). IRAK1 antibody (Proteintech 10478-2-AP) staining in (E, F) BMDMs and (G) Irak1 −/− BMDMs on co-stimulation with P3C and Kdo-2 Lipid A (500 nM each). Scale Bar: 10 μ . (Panel F) Tamhane’s T2 multiple comparisons test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments. See also supporting , Movies 1-5.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Infection, Expressing, Staining

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) Time course of IRAK1 clustering on multi-PRR stimulation with Salmonella typhimurium with the color bands showing the range. Three fields were imaged. (B-J) Kdo-2 Lipid A and P3C co-treatment (50 nM) in iBMDMs. (B) Percentage of iBMDMs showing IRAK1 clustering at 90 min, (C) IRAK1 clustering intensity in iBMDMs on single vs co-TLR stimulation at 90 min, (D) Time course of IRAK1 clustering. (E) Images of IRAK1mCherry and IRAK1 antibody staining in iBMDMs. Scale Bar: 10 μ . (F) IRAK1 antibody (SCBT sc-5288) staining in Irak1 −/− iBMDMs. Scale Bar: 10 μ . (G) Cytofluorogram for IRAK1 antibody (SCBT sc-5288) and IRAK1mCherry. (H) Independent Component Analysis for signals from IRAK1 antibody (SCBT sc-5288) and IRAK1 mCherry. (I) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of IRAK1 antibody (CST 4504S) staining in IRAK1 mCherry clusters in iBMDMs. (J) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of IRAK1 antibody (CST 4504S) staining in IRAK1 mCherry clusters in iBMDMs. (K) Time course of IRAK1 clustering on co-stimulation of TLR4 and TLR2 with soluble biglycan (1 μ g/mL), a DAMP or co-TLR stimulation with the PAMPs - P3C and Kdo-2 Lipid A (50 nM each). Data are represented as median. (Panels B, C) Kolmogorov-Smirnov test. (Panels I, J) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) Co-staining of IRAK1 clusters and proteasome in iBMDMs. Kdo-2 Lipid A and P3C co-treatment (50 nM). Scale Bar: 10 μ . (B) Dose response of IRAK1 clustering in iBMDMs with Kdo-2 Lipid A and P3C ± 2h pre-treatment with 4uL of monensin, BD GolgiStop™ for every 3 mL of cell culture. (C-K) Kdo-2 Lipid A and P3C co-treatment (50 nM) in iBMDMs. Co-staining of IRAK1 clusters and (C) MyD88, (D) TICAM2, (E) IRAK4, (F) IRAK2, (G) TRAF6 and (H) pTBK1. Scale Bar: 10 μ . (I) Summary of IRAK1 clustering with TLR signaling components. ND: Not Determined. (J-L) IRAK1 clustering in iBMDMs 0, 2 and 3h post-treatment with 50 nM Kdo-2 Lipid A and P3C ± 30 min pre-treatment with (J) dynasore, an internalization inhibitor (20 μ M); (K) ST2825, a MyD88 dimerization inhibitor (20 μ M); and (L) thymoquinone, an IRAK1 kinase inhibitor (25 μ M). (Panels B, J-L) Data are represented as median ± MAD. (Panels J-L) Unpaired t test with Holm-Šídák’s correction. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments. See also supporting .

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Cell Culture

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A-K) Kdo-2 Lipid A and P3C co-treatment (50 nM each) in iBMDMs. Co-staining of IRAK1 clusters and (A) ßTrCp, (B) pellino, and (C) IRAK3. Scale Bar: 10 μ . (D) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of TRAF6 antibody staining in IRAK1 clusters. (E) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of TRAF6 antibody staining in IRAK1 clusters in iBMDMs. (F) PCC and (G) MCC for pTBK1 antibody staining in IRAK1 clusters. (H-K) Proximity ligation assay in iBMDMs. (H) IRAK1-TRAF6 PLA, (I) IRAK1-pTBK1 PLA, (J) IRAK2-TRAF6 PLA, (K) IRAK1-pTBK1 PLA. (L, M) Single or co-TLR stimulation of TLR4 and TLR1/2 with Kdo-2 Lipid A and P3C (50 nM each). Quantification of (L) pp65 nuclear translocation (M) pATF2 nuclear translocation. (N) Quantification of pATF2 nuclear translocation on co-TLR stimulation of TLR4 and TLR1/2 with Kdo-2 Lipid A and P3C ± 30 min pre-treatment with dynasore, an internalization inhibitor, 20 μ M, ST2825, a MyD88 dimerization inhibitor, 20 μ M, and thymoquinone, an IRAK1 kinase inhibitor, 25 μ M. (D-G) Data are represented as mean ± SD. (H-N) Data are represented as median ± MAD. (Panels D-G) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. (Panels H-M) Tukey’s multiple comparisons test. (Panel N) Dunnett’s multiple comparisons test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Proximity Ligation Assay, Translocation Assay

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: Kdo-2 Lipid A and P3C co-treatment (50 nM) in iBMDMs. (A) Co-staining of IRAK1 clusters and ASC (SCBT sc-22514-R). Scale Bar: 10 μ . (B) ASC (SCBT sc-22514-R) staining in Irak1 −/− iBMDMs. Scale Bar: 10 μ . (C) Quantification of ASC (SCBT sc-22514-R) clustering in iBMDMs after pairwise stimulation with TLR ligands – Kdo-2 Lipid A (0.5, 50 nM) and P3C (0.5, 50 nM). Data are represented as mean (n = 3). IRAK1 and ASC cluster correlation calculated using IRAK1 clustering data from . (D, E) Proximity ligation assays in iBMDMs. Data are represented as Median ± SD. (D) IRAK1-ASC PLA, (E) IRAK1-NLRP3 PLA. (F) Single cell live imaging of IRAK1 clustering and ASC clustering in IRAK1mCh ASC GFP iBMDMs on 2h priming with Kdo-2 Lipid A and P3C (50 nM) followed by Nigericin (10 μ M) trigger for 2h. The cell tracks could be broadly divided into three groups. Group 1 cells showing ASC specks exhibited IRAK1 clusters for a comparable time period prior to ASC speck formation. The non-ASC specking cells could be divided into two groups - group 2 cells had the largest IRAK1 clusters throughout the time course, while group 3 cells had either very weak or undetectable IRAK1 clusters. (G) ASC specking in IRAK1-mCh ASC-GFP +/− thymoquinone, an IRAK1 kinase inhibitor (25 μ M) or Irak1 −/− ASC-GFP iBMDMs on 2h priming with Kdo-2 Lipid A and P3C (50 nM) followed by Nigericin (10 μ M) trigger for 2h. (Panel C) Paired t test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments. See also supporting , Movies 6-8.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Ligation, Imaging

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: Kdo-2 Lipid A and P3C co-treatment (50 nM each) in iBMDMs. (A) Cytofluorogram for ASC antibody (SCBT sc-22514-R) and IRAK1mCherry. (B) Independent Component Analysis for signals from ASC antibody (SCBT sc-22514-R) and IRAK1 mCherry. (C) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of ASC antibody (SCBT sc-22514-R) staining in IRAK1 clusters. (D) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of ASC antibody (SCBT sc-22514-R) staining in IRAK1 clusters in iBMDMs. (E) Co-staining of IRAK1 clusters and pASC. (F) pASC staining in Irak1 −/− iBMDMs. (G) PCC and (H) MCC for pASC antibody staining in IRAK1 clusters. (I) Co-staining of IRAK1 clusters and NLRP3. (J) NLRP3 staining in Irak1 −/− iBMDMs. (K) PCC and (L) MCC for NLRP3 antibody staining in IRAK1 clusters. (M) Co-staining of IRAK1 clusters and NLRC4. (N) NLRC4 staining in Irak1 −/− iBMDMs. (O) PCC and (P) MCC for NLRC4 antibody staining in IRAK1 clusters. (Panels C, D, G, H, K, L, O, P) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. Automated imaging of at least 2 (Panels F, J, N) fields of cells. Data are represented as mean ± SD. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Imaging

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A-D) Proximity ligation assay in BMDMs. At least 20,000 cells imaged for all graphs. Percentage of cells showing PLA spots stated at the base of the graphs. Co-TLR stimulation with Kdo-2 Lipid A and P3C co-treatment (500 nM each). Multi-PRR stimulation using heat-killed Yersinia pseudotuberculosis (MOI 100). (A) IRAK1-ASC PLA intensity, (B) IRAK1-ASC PLA spot area, (C) IRAK1-NLRP3 PLA intensity, (D) IRAK1-NLRP3 PLA spot area. (E-G) Cytokine secretion measured by ELISA in BMDMs primed with single TLR ligands: Kdo-2 Lipid A or P3C (1 uM each) or with co-TLR stimulation using 500 nM of both ligands, followed by ATP (5 mM) trigger for 2h. Mean ± SD from at least 4 wells. (E) IL1 α , (F) IL1 β and (G) TNF. (H) qPCR quantification of Pycard (ASC) , Nlrp3 , Casp1 , IL18 , IL1a and IL1b in BMDMs primed with heat-killed Yersinia pseudotuberculosis (MOI 100) or with co-TLR stimulation using 500 nM of both Kdo-2 Lipid A and P3C. Mean ± SD from at least 4 wells. (I-K) Cytokine secretion measured as detailed in E-G in BMDMs pretreated with thymoquinone, an IRAK1 kinase inhibitor (25 μ M). (I) IL1 α , (J) IL1 β and (K) TNF. (Panels A-D) Kolmogorov-Smirnov test. (Panels E-G, K-M) Tukey’s multiple comparisons test. (Panels H-J) Two-way ANOVA with Geisser-Greenhouse correction and Šídák’s multiple comparison test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). See also supporting .

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Proximity Ligation Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: Proximity ligation assay in BMDMs. At least 20,000 cells imaged for all graphs. Co-TLR stimulation with Kdo-2 Lipid A and P3C co-treatment (500 nM). Multi-PRR stimulation using heat-killed Yersinia pseudotuberculosis (MOI 100). (A) IRAK1-pASC PLA intensity, (B) IRAK1-pASC PLA spot area. (C-E) Co-TLR (PAMP: Kdo-2 Lipid A and P3C, DAMP: soluble biglycan) or multi-PRR priming (heat-killed Yersinia pseudotuberculosis , MOI 100) followed by Nigericin trigger (10 μ M) for 2h (C) IRAK1-ASC PLA, (D) IRAK1-pASC PLA, (E) IRAK1-NLRP3 PLA. qPCR quantification of (F) IL1a and (G) IL1b in BMDMs primed with co-TLR stimulation using 500 nM of both Kdo-2 Lipid A and P3C or single-TLR stimulation with either 1 μ M Kdo-2 Lipid A or 1 μ M P3C or with heat-killed Mean ± SD from at least 4 wells. (Panels A-B) Kolmogorov-Smirnov test. (Panels F, G) Šídák’s multiple comparisons test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Proximity Ligation Assay

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A) IRAK1 clustering in iBMDMs 0, 2 and 3h post-treatment with 50 nM Kdo-2Lipid A and P3C ± 30 min pre-treatment with JNK inhibitor (10 μ M). (B) Co-staining of IRAK1 clusters and pJNK in iBMDMs. Kdo-2 Lipid A and P3C co-treatment (50 nM). Scale Bar: 10 μ . (C, D) Proximity ligation assay in BMDMs. At least 20,000 cells imaged for all graphs. Co-TLR stimulation with Kdo-2 Lipid A and P3C co-treatment (500 nM). (C) IRAK1-pJNK PLA intensity, (D) IRAK1-pJNK PLA spot area. (E-G) Cytokine secretion measured by ELISA in BMDMs pretreated with JNK inhibitor, 10 μ M, primed with single TLR ligands: Kdo-2 Lipid A or P3C (1 uM each) or with co-TLR stimulation using 500 nM of both ligands, followed by ATP (5 mM) trigger for 2h. (E) IL1 α , (F) IL1 β and (G) TNF. (H) Co-staining of IRAK1 clusters and pMKK7 in iBMDMs. Kdo-2 Lipid A and P3C co-treatment (50 nM). Scale Bar: 10 μ . (I,J) Fluorescence lifetime measurement of ASC where the acceptor/donor pair was pJNK/ASC primed with multi-PRR stimulation by heat-killed Yersinia pseudotuberculosis or Pseudomonas aeruginosa (MOI 100) in WT and Irak1 −/− BMDMs. (Panel A) Unpaired t test with Holm-Šídák’s correction. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). (Panels C,D) Kolmogorov-Smirnov test. (Panels E-G) Tukey’s multiple comparisons test. (Panel J) Two-way ANOVA and Šídák’s multiple comparison test. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments. See also supporting .

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Proximity Ligation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: bioRxiv

Article Title: IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

doi: 10.1101/2019.12.26.888776

Figure Lengend Snippet: (A-C, E-G, I) Kdo-2 Lipid A and P3C co-treatment (50 nM each) in iBMDMs. (D, H) Co-TLR (PAMP: Kdo-2 Lipid A and P3C, 50 nM each) or multi-PRR priming (heat-killed Yersinia pseudotuberculosis , MOI 100) followed by Nigericin (10 μ M) trigger for 2h. (A) Quantification of the Pearson’s correlation coefficient (PCC) for the correlation of pJNK antibody staining in IRAK1 clusters. (B) Quantification of the Mander’s colocalization coefficient (MCC) for the co-occurrence of pJNK antibody staining in IRAK1 clusters. (C) pJNK staining in Irak1 −/− iBMDMs. (D) IRAK1-pJNK PLA. (E) PCC and (F) MCC for pMKK7 antibody staining in IRAK1 clusters. (G) pMKK7 staining in Irak1 −/− iBMDMs. (H) IRAK1-pMKK7 PLA in BMDMs. (I) pMKK4 staining in iBMDMs mIRAK1mCherry. (Panels A, B, E, F) Each data point represents value from a randomly selected non-over lapping field of cells. Paired t test. Data are represented as mean ± SD. Automated imaging of at least 2 (Panels C, G, I) fields of cells. At least 20000 (Panels D, H) cells imaged. p = 0.1234 (ns), 0.0332(*); 0.0021 (**); 0.0002 (***); < 0.0001 (****). Data shown are representative of at least two independent experiments.

Article Snippet: Antibody pairs used for PLA in BMDMs: IRAK1 Rb (Proteintech 10478-2-AP) and ASC Ms (SCBT sc-514414); IRAK1 Rb (Proteintech 10478-2-AP) and NLRP3 Ms (AdipoGen AG-20B-0014-C100); IRAK1 Ms (SCBT sc-5288) and pASC Rb (ECM Biosciences AP5631); IRAK1 Ms (SCBT sc-5288) and pJNK Rb (Invitrogen 700031); IRAK1 Ms (SCBT sc-5288) and pMKK7 Rb (CST 4171S).

Techniques: Staining, Imaging