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rabbit anti human myd88  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit anti human myd88
a Schematic representation of TLR4 signaling, ligands used and contact point of the <t>MyD88</t> inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.
Rabbit Anti Human Myd88, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti-human+myd88/MyD88+Rabbit+mAb/pmc11558003-380-13-17
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rabbit anti human  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc rabbit anti human
a Schematic representation of TLR4 signaling, ligands used and contact point of the <t>MyD88</t> inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.
Rabbit Anti Human, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human mouse rat monkey myd88  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc human mouse rat monkey myd88
Figure 2. IFN-I priming by the gut microbiota involves the cytosolic STING pathway (A) Basal Ifnb1 response in Ifnb+/Db-luc mice and representative images before and after 2 weeks oral antibiotics. Data are means ± SEM (n = 14), p value determined by two-tailed paired t test. (B) RNA-seq heatmap showing relative expression levels of selected interferon-stimulated genes (ISGs) in colon of WT (n = 4) or MTIS (n = 3) mice treated (or not) with antibiotics. (C) Overview of Ifnb1 luciferase reporter mouse strains. (D) Immunoblots of STING, TRIF, and <t>MYD88</t> in indicated mice.
Human Mouse Rat Monkey Myd88, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit  (Cell Signaling Technology Inc)
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Cell Signaling Technology Inc rabbit
Figure 2. IFN-I priming by the gut microbiota involves the cytosolic STING pathway (A) Basal Ifnb1 response in Ifnb+/Db-luc mice and representative images before and after 2 weeks oral antibiotics. Data are means ± SEM (n = 14), p value determined by two-tailed paired t test. (B) RNA-seq heatmap showing relative expression levels of selected interferon-stimulated genes (ISGs) in colon of WT (n = 4) or MTIS (n = 3) mice treated (or not) with antibiotics. (C) Overview of Ifnb1 luciferase reporter mouse strains. (D) Immunoblots of STING, TRIF, and <t>MYD88</t> in indicated mice.
Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti-human+myd88/MyD88+Rabbit+mAb/pmc09108609-231-3-9
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rabbit anti human myd88  (Proteintech)
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Proteintech rabbit anti human myd88
Protein expression of ERβ, TLR4 and the downstream molecules <t>myd88</t> and MMP2 in NSCLC cell lines treated with estrogen and inhibitors. a , b Western blot analysis of ERβ, TLR4, myd88 and MMP2 protein levels in A549 ( a ) and H1793 ( b ) cells treated with DMSO (CTRL), E2 (10 nM), E2 + Ful (100 nM), E2 + CLI-095 (100 nM), or E2 + Ful + CLI-095 for 48 h. The combination of fulvestrant and CLI-095 synergistically inhibited the ERβ and TLR4/myd88/MMP2 pathway. c , d Detection of the relative O.D. values of the Western blot analysis. e Immunofluorescence staining of ERβ and TLR4 in A549 cells. Scale bar, 200 μm. f Detection of the mean intensity of fluorescence staining
Rabbit Anti Human Myd88, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-human myd88 (1:100 dilution)  (Enzo Biochem)
90
Enzo Biochem rabbit anti-human myd88 (1:100 dilution)
Protein expression of ERβ, TLR4 and the downstream molecules <t>myd88</t> and MMP2 in NSCLC cell lines treated with estrogen and inhibitors. a , b Western blot analysis of ERβ, TLR4, myd88 and MMP2 protein levels in A549 ( a ) and H1793 ( b ) cells treated with DMSO (CTRL), E2 (10 nM), E2 + Ful (100 nM), E2 + CLI-095 (100 nM), or E2 + Ful + CLI-095 for 48 h. The combination of fulvestrant and CLI-095 synergistically inhibited the ERβ and TLR4/myd88/MMP2 pathway. c , d Detection of the relative O.D. values of the Western blot analysis. e Immunofluorescence staining of ERβ and TLR4 in A549 cells. Scale bar, 200 μm. f Detection of the mean intensity of fluorescence staining
Rabbit Anti Human Myd88 (1:100 Dilution), supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Article Snippet: The following antibodies were used: rabbit anti-human CD14 (1:1000, Cell Signaling, D7A2T, #56082), rabbit anti-human MyD88 (1:1000, Cell Signaling, D80F5, #4283), rabbit anti-human TRIF (1:1000, Cell Signaling, #4596), mouse anti-human TICAM-2/TRAM (1:500, Bio Legend, #659402), rabbit anti-human TLR4 (1:1000, Cell Signaling, E5D8T, #38519), mouse anti-human β-actin (1:1000, LI-COR Biosciences, #926-42212), goat anti-rabbit HRP-linked (1:3000, BIO-RAD, #1706515), goat anti-mouse IRDye 680RD-linked (1:15000, LI-COR Biosciences, #926-68070), horse anti-mouse HRP-linked (1:2000, Cell Signaling, #7076), goat anti-mouse HRP-linked (1:2000, Cell Signaling, #7074).

Techniques: Immunofluorescence, Microscopy, Transfection, Construct

Figure 2. IFN-I priming by the gut microbiota involves the cytosolic STING pathway (A) Basal Ifnb1 response in Ifnb+/Db-luc mice and representative images before and after 2 weeks oral antibiotics. Data are means ± SEM (n = 14), p value determined by two-tailed paired t test. (B) RNA-seq heatmap showing relative expression levels of selected interferon-stimulated genes (ISGs) in colon of WT (n = 4) or MTIS (n = 3) mice treated (or not) with antibiotics. (C) Overview of Ifnb1 luciferase reporter mouse strains. (D) Immunoblots of STING, TRIF, and MYD88 in indicated mice.

Journal: Immunity

Article Title: The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis.

doi: 10.1016/j.immuni.2022.04.006

Figure Lengend Snippet: Figure 2. IFN-I priming by the gut microbiota involves the cytosolic STING pathway (A) Basal Ifnb1 response in Ifnb+/Db-luc mice and representative images before and after 2 weeks oral antibiotics. Data are means ± SEM (n = 14), p value determined by two-tailed paired t test. (B) RNA-seq heatmap showing relative expression levels of selected interferon-stimulated genes (ISGs) in colon of WT (n = 4) or MTIS (n = 3) mice treated (or not) with antibiotics. (C) Overview of Ifnb1 luciferase reporter mouse strains. (D) Immunoblots of STING, TRIF, and MYD88 in indicated mice.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-human/mouse/rat IRF3; D83B9 Cell Signaling Technology #4302; RRID: AB_1904036 Rabbit anti-human/mouse phospho-IRF3 (Ser396); 4D4G Cell Signaling Technology #4947; RRID: AB_823547 Rabbit anti-human/mouse/rat phospho-STAT1 (Tyr701); D4A7 Cell Signaling Technology #7649; RRID: AB_10950970 Rabbit anti-mouse phospho-STING (Ser365); D8F4W Cell Signaling Technology #72971; RRID: AB_279983 Rabbit anti-human/mouse STING; D2P2F Cell Signaling Technology #13647; RRID: AB_2732796 Rabbit anti-human/mouse/rat/monkey MYD88; D80F5 Cell Signaling Technology #4283; RRID: AB_10547882 Horse anti-mouse HRP-linked IgG Cell Signaling Technology #7076; RRID: AB_330924 Goat anti-rabbit HRP-linked IgG Cell Signaling Technology #7074; RRID: AB_2099233 Rabbit anti-mouse/rat/human NAK (TBK1); EP611Y Abcam #ab40676; RRID: AB_776632 Rabbit anti-mouse/rat/human NAK/TBK1 (phospho S172); EPR2867(2) Abcam #ab109272; RRID: AB_10862438 Mouse anti-mouse/rat/human STAT1a p91; C-111 Santa Cruz Biotechnologies #sc-417; RRID: AB_675902 Rabbit anti-TRIF/TICAM1 Proteintech #23288-1-AP; RRID: AB_2879247 Bacterial and virus strains Escherichia coli MC1061 (araD139 D(ara, leu) 7697 DlacX74 galU galK hsr hsm+ strA) Casadaban et al., 1980 Provided by Sun Nyunt Wai, Umeå University N/A Escherichia coli MC1061 DlpxM (derivative of MC1061) This paper N/A Vibrio cholerae V:5/04 This paper N/A Vibrio cholerae V:5/04 Dvcc This paper N/A Enterococcus faecalis strain OG1RF Dunny et al., 1978 ATCC ATCC: 47077 Bacteroides fragilis strain NCTC9343 DSMZ-German Collection of Microorganisms and Cell Cultures GmbH DSM No. 2151 Herpes simplex virus type I strain KOS KOS/Dlux/OriL (HSV-I-luc) containing a firefly luciferase reporter (Summers and Leib, 2002) N/A Vesicular stomatitis virus VSV-AV2 (attenuated strain) (Stojdl et al., 2003) Provided by Kröger A, HZI, Germany N/A Vesicular stomatitis virus VSV-GFP (virulent) (Nair et al., 2014) Provided by Kröger A, HZI, Germany N/A Chemicals, peptides, and recombinant proteins Blasticidin InvivoGen #ant-bl-1 Puromycin InvivoGen #ant-pr-1 Gentamicin solution Sigma-Aldrich (Merck) #G1397 Iscove’s Modified Dulbecco’s Medium (IMDM Gibco (Thermo Fisher Scientific) #31980-22 Roswell Park Memorial Institute (RPMI) 1640 medium Gibco (Thermo Fisher Scientific) #72400013 (Continued on next page) e1 Immunity 55, 847–861.e1–e10, May 10, 2022

Techniques: Two Tailed Test, RNA Sequencing, Expressing, Luciferase, Western Blot

Figure 4. Bacterial MVs prime the STING-IFN-I axis to promote antiviral immunity (A) Layout of experiments in (B) to (D). (B–D) HSV-1 luciferase level in (B) and (C) WT mice or (D) Myd88/Ticam1/Mavs/ mice inoculated (or not) with either (B) live (2 3 108) E. coli WT or DlpxM or (C and D) 25 mg MVs and allowed to recover for 2 weeks prior to intraperitoneal HSV-1-luc infection. The rainbow scales indicate p/s/cm2/sr. Data in (B), (C) (n = 3–5), and D (n = 8) are means ± SEM (from two experiments). p values determined by one-way ANOVA with Bonferroni post-test. See also Figure S5.

Journal: Immunity

Article Title: The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis.

doi: 10.1016/j.immuni.2022.04.006

Figure Lengend Snippet: Figure 4. Bacterial MVs prime the STING-IFN-I axis to promote antiviral immunity (A) Layout of experiments in (B) to (D). (B–D) HSV-1 luciferase level in (B) and (C) WT mice or (D) Myd88/Ticam1/Mavs/ mice inoculated (or not) with either (B) live (2 3 108) E. coli WT or DlpxM or (C and D) 25 mg MVs and allowed to recover for 2 weeks prior to intraperitoneal HSV-1-luc infection. The rainbow scales indicate p/s/cm2/sr. Data in (B), (C) (n = 3–5), and D (n = 8) are means ± SEM (from two experiments). p values determined by one-way ANOVA with Bonferroni post-test. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-human/mouse/rat IRF3; D83B9 Cell Signaling Technology #4302; RRID: AB_1904036 Rabbit anti-human/mouse phospho-IRF3 (Ser396); 4D4G Cell Signaling Technology #4947; RRID: AB_823547 Rabbit anti-human/mouse/rat phospho-STAT1 (Tyr701); D4A7 Cell Signaling Technology #7649; RRID: AB_10950970 Rabbit anti-mouse phospho-STING (Ser365); D8F4W Cell Signaling Technology #72971; RRID: AB_279983 Rabbit anti-human/mouse STING; D2P2F Cell Signaling Technology #13647; RRID: AB_2732796 Rabbit anti-human/mouse/rat/monkey MYD88; D80F5 Cell Signaling Technology #4283; RRID: AB_10547882 Horse anti-mouse HRP-linked IgG Cell Signaling Technology #7076; RRID: AB_330924 Goat anti-rabbit HRP-linked IgG Cell Signaling Technology #7074; RRID: AB_2099233 Rabbit anti-mouse/rat/human NAK (TBK1); EP611Y Abcam #ab40676; RRID: AB_776632 Rabbit anti-mouse/rat/human NAK/TBK1 (phospho S172); EPR2867(2) Abcam #ab109272; RRID: AB_10862438 Mouse anti-mouse/rat/human STAT1a p91; C-111 Santa Cruz Biotechnologies #sc-417; RRID: AB_675902 Rabbit anti-TRIF/TICAM1 Proteintech #23288-1-AP; RRID: AB_2879247 Bacterial and virus strains Escherichia coli MC1061 (araD139 D(ara, leu) 7697 DlacX74 galU galK hsr hsm+ strA) Casadaban et al., 1980 Provided by Sun Nyunt Wai, Umeå University N/A Escherichia coli MC1061 DlpxM (derivative of MC1061) This paper N/A Vibrio cholerae V:5/04 This paper N/A Vibrio cholerae V:5/04 Dvcc This paper N/A Enterococcus faecalis strain OG1RF Dunny et al., 1978 ATCC ATCC: 47077 Bacteroides fragilis strain NCTC9343 DSMZ-German Collection of Microorganisms and Cell Cultures GmbH DSM No. 2151 Herpes simplex virus type I strain KOS KOS/Dlux/OriL (HSV-I-luc) containing a firefly luciferase reporter (Summers and Leib, 2002) N/A Vesicular stomatitis virus VSV-AV2 (attenuated strain) (Stojdl et al., 2003) Provided by Kröger A, HZI, Germany N/A Vesicular stomatitis virus VSV-GFP (virulent) (Nair et al., 2014) Provided by Kröger A, HZI, Germany N/A Chemicals, peptides, and recombinant proteins Blasticidin InvivoGen #ant-bl-1 Puromycin InvivoGen #ant-pr-1 Gentamicin solution Sigma-Aldrich (Merck) #G1397 Iscove’s Modified Dulbecco’s Medium (IMDM Gibco (Thermo Fisher Scientific) #31980-22 Roswell Park Memorial Institute (RPMI) 1640 medium Gibco (Thermo Fisher Scientific) #72400013 (Continued on next page) e1 Immunity 55, 847–861.e1–e10, May 10, 2022

Techniques: Luciferase, Infection

Figure 5. Bacterial MVs encase and deliver DNA into the host cytosol to activate cGAS-STING-IFN-I signaling (A) Atomic force microscopic images of membrane vesicles from V. cholerae Dvcc and E. coli WT (image section: 0.25 3 0.5 mm). (B) V. cholerae Dvcc MVs or E. coli DlpxM MVs, left untreated (i) or treated with benzonase to strip them off extravesicular nucleic acids (ii) or lysed before benzonase treatment (iii), were subjected to nucleic acid extraction, and extracts were analyzed by agarose gel separation. One representative out of three experiments shown. (C and D) Relative mRNA levels of Ifnb1 in Myd88/Ticam1/Mavs/ BMDMs stimulated (or not) with benzonase-treated V. cholerae Dvcc MVs (C) or E. coli DlpxM MVs (D) for 6 h. Data are means ± SEM of 3 independent experiments. (E and F) 50 mg mL1 V. cholerae Dvcc MV-induced Mx2 luciferase activity in HEK293 cells transfected with cGAS (pBabe-cGAS) or control vector (pBabe-empty). (F) 25 mg mL1 E. coli DlpxM MV-induced Mx2 luciferase activity in HEK293 cells transfected with cGAS or control vector. Data in (E) and (F) are means ± SEM of 3 independent experiments. p values determined by one-way ANOVA with Bonferroni post-test. (G and H) Ifnb1 transcript levels in WT and Cgas/ BMDMs (G) cocultured with E. coli DlpxM for 3 h in transwells to prevent direct bacteria-host cell contact or (H) incubated for 6 h with 25 mg mL1 E. coli DlpxM MVs. Data are means ± SEM of 3 independent experiments. (I) Proposed model for extracellular bacteria-mediated activation of the cGAS-STING-IFN-I axis. See also Figure S6.

Journal: Immunity

Article Title: The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis.

doi: 10.1016/j.immuni.2022.04.006

Figure Lengend Snippet: Figure 5. Bacterial MVs encase and deliver DNA into the host cytosol to activate cGAS-STING-IFN-I signaling (A) Atomic force microscopic images of membrane vesicles from V. cholerae Dvcc and E. coli WT (image section: 0.25 3 0.5 mm). (B) V. cholerae Dvcc MVs or E. coli DlpxM MVs, left untreated (i) or treated with benzonase to strip them off extravesicular nucleic acids (ii) or lysed before benzonase treatment (iii), were subjected to nucleic acid extraction, and extracts were analyzed by agarose gel separation. One representative out of three experiments shown. (C and D) Relative mRNA levels of Ifnb1 in Myd88/Ticam1/Mavs/ BMDMs stimulated (or not) with benzonase-treated V. cholerae Dvcc MVs (C) or E. coli DlpxM MVs (D) for 6 h. Data are means ± SEM of 3 independent experiments. (E and F) 50 mg mL1 V. cholerae Dvcc MV-induced Mx2 luciferase activity in HEK293 cells transfected with cGAS (pBabe-cGAS) or control vector (pBabe-empty). (F) 25 mg mL1 E. coli DlpxM MV-induced Mx2 luciferase activity in HEK293 cells transfected with cGAS or control vector. Data in (E) and (F) are means ± SEM of 3 independent experiments. p values determined by one-way ANOVA with Bonferroni post-test. (G and H) Ifnb1 transcript levels in WT and Cgas/ BMDMs (G) cocultured with E. coli DlpxM for 3 h in transwells to prevent direct bacteria-host cell contact or (H) incubated for 6 h with 25 mg mL1 E. coli DlpxM MVs. Data are means ± SEM of 3 independent experiments. (I) Proposed model for extracellular bacteria-mediated activation of the cGAS-STING-IFN-I axis. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-human/mouse/rat IRF3; D83B9 Cell Signaling Technology #4302; RRID: AB_1904036 Rabbit anti-human/mouse phospho-IRF3 (Ser396); 4D4G Cell Signaling Technology #4947; RRID: AB_823547 Rabbit anti-human/mouse/rat phospho-STAT1 (Tyr701); D4A7 Cell Signaling Technology #7649; RRID: AB_10950970 Rabbit anti-mouse phospho-STING (Ser365); D8F4W Cell Signaling Technology #72971; RRID: AB_279983 Rabbit anti-human/mouse STING; D2P2F Cell Signaling Technology #13647; RRID: AB_2732796 Rabbit anti-human/mouse/rat/monkey MYD88; D80F5 Cell Signaling Technology #4283; RRID: AB_10547882 Horse anti-mouse HRP-linked IgG Cell Signaling Technology #7076; RRID: AB_330924 Goat anti-rabbit HRP-linked IgG Cell Signaling Technology #7074; RRID: AB_2099233 Rabbit anti-mouse/rat/human NAK (TBK1); EP611Y Abcam #ab40676; RRID: AB_776632 Rabbit anti-mouse/rat/human NAK/TBK1 (phospho S172); EPR2867(2) Abcam #ab109272; RRID: AB_10862438 Mouse anti-mouse/rat/human STAT1a p91; C-111 Santa Cruz Biotechnologies #sc-417; RRID: AB_675902 Rabbit anti-TRIF/TICAM1 Proteintech #23288-1-AP; RRID: AB_2879247 Bacterial and virus strains Escherichia coli MC1061 (araD139 D(ara, leu) 7697 DlacX74 galU galK hsr hsm+ strA) Casadaban et al., 1980 Provided by Sun Nyunt Wai, Umeå University N/A Escherichia coli MC1061 DlpxM (derivative of MC1061) This paper N/A Vibrio cholerae V:5/04 This paper N/A Vibrio cholerae V:5/04 Dvcc This paper N/A Enterococcus faecalis strain OG1RF Dunny et al., 1978 ATCC ATCC: 47077 Bacteroides fragilis strain NCTC9343 DSMZ-German Collection of Microorganisms and Cell Cultures GmbH DSM No. 2151 Herpes simplex virus type I strain KOS KOS/Dlux/OriL (HSV-I-luc) containing a firefly luciferase reporter (Summers and Leib, 2002) N/A Vesicular stomatitis virus VSV-AV2 (attenuated strain) (Stojdl et al., 2003) Provided by Kröger A, HZI, Germany N/A Vesicular stomatitis virus VSV-GFP (virulent) (Nair et al., 2014) Provided by Kröger A, HZI, Germany N/A Chemicals, peptides, and recombinant proteins Blasticidin InvivoGen #ant-bl-1 Puromycin InvivoGen #ant-pr-1 Gentamicin solution Sigma-Aldrich (Merck) #G1397 Iscove’s Modified Dulbecco’s Medium (IMDM Gibco (Thermo Fisher Scientific) #31980-22 Roswell Park Memorial Institute (RPMI) 1640 medium Gibco (Thermo Fisher Scientific) #72400013 (Continued on next page) e1 Immunity 55, 847–861.e1–e10, May 10, 2022

Techniques: Membrane, Stripping Membranes, Extraction, Agarose Gel Electrophoresis, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Bacteria, Incubation, Activation Assay

Protein expression of ERβ, TLR4 and the downstream molecules myd88 and MMP2 in NSCLC cell lines treated with estrogen and inhibitors. a , b Western blot analysis of ERβ, TLR4, myd88 and MMP2 protein levels in A549 ( a ) and H1793 ( b ) cells treated with DMSO (CTRL), E2 (10 nM), E2 + Ful (100 nM), E2 + CLI-095 (100 nM), or E2 + Ful + CLI-095 for 48 h. The combination of fulvestrant and CLI-095 synergistically inhibited the ERβ and TLR4/myd88/MMP2 pathway. c , d Detection of the relative O.D. values of the Western blot analysis. e Immunofluorescence staining of ERβ and TLR4 in A549 cells. Scale bar, 200 μm. f Detection of the mean intensity of fluorescence staining

Journal: Clinical & Translational Oncology

Article Title: Targeting Toll-like receptor 4 with CLI-095 (TAK-242) enhances the antimetastatic effect of the estrogen receptor antagonist fulvestrant on non-small cell lung cancer

doi: 10.1007/s12094-020-02353-3

Figure Lengend Snippet: Protein expression of ERβ, TLR4 and the downstream molecules myd88 and MMP2 in NSCLC cell lines treated with estrogen and inhibitors. a , b Western blot analysis of ERβ, TLR4, myd88 and MMP2 protein levels in A549 ( a ) and H1793 ( b ) cells treated with DMSO (CTRL), E2 (10 nM), E2 + Ful (100 nM), E2 + CLI-095 (100 nM), or E2 + Ful + CLI-095 for 48 h. The combination of fulvestrant and CLI-095 synergistically inhibited the ERβ and TLR4/myd88/MMP2 pathway. c , d Detection of the relative O.D. values of the Western blot analysis. e Immunofluorescence staining of ERβ and TLR4 in A549 cells. Scale bar, 200 μm. f Detection of the mean intensity of fluorescence staining

Article Snippet: The primary antibodies used for the Western blot analyses and Immunofluorescence included rabbit anti-human ERβ (1:1,000) from Abcam (Cat: ab3577), rabbit anti-human TLR4 (1:1,000) from Abcam (Cat: ab13556), rabbit anti-human myd88 (1:1,000) from Proteintech (Cat: 23230-1-AP), and mouse anti-human GAPDH (1:10,000) from Cell Signaling Technology (CST; Cat: 51332; USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence