rabbit anti β actin monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti β actin monoclonal antibody
    Rabbit Anti β Actin Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti ß actin monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti ß actin monoclonal antibody
    Rabbit Anti ß Actin Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti β actin monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti β actin monoclonal antibody
    Rabbit Anti β Actin Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti β actin monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti β actin monoclonal antibody
    Rabbit Anti β Actin Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibody against β actin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibody against β actin
    Antibody Against β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti α tubulin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti α tubulin
    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised <t>to</t> <t>α-tubulin</t> and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
    Anti α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
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    anti α tubulin - by Bioz Stars, 2023-06
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    1) Product Images from "FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins"

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202201735

    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
    Figure Legend Snippet: (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Techniques Used: Genome Wide, CRISPR, DNA Sequencing, Sequencing, Quantitative RT-PCR, Western Blot, Microscopy, Live Cell Imaging

    (A) Live cell imaging showing the percentage of Draq7-positive (dead) WT and C5aR1 −/− macrophages to PBS or PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. ** = P < 0.01 for WT versus C5aR1 −/− at 15 h post toxin treatment; by unpaired t test. (B) Flow cytometric analysis and median fluorescence intensity of C5aR1 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, red and blue lines represent FBXO11 −/− (E2 and E3, respectively), and dark grey line represents C5aR1 −/− macrophages. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Confocal microscopy images of WT, FBXO11 −/− , and C5aR1 −/− macrophages. PFA-fixed cells were stained using anti-C5aR1 (green), and nuclei were stained with DAPI (blue). Scale bar corresponds to 100 μM. (D) Immunoblot analysis of C5aR1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of C5aR1 in WT macrophages that were treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophage. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (F) qRT-PCR analysis of C5aR1 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. C5aR1 levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (G) Flow cytometric analysis and MFI of recombinant LukS-PV subunit binding to WT and FBXO11 −/− macrophages. Grey line represents isotype, dotted black line represents no LukS-PV negative control, solid black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages treated with LukS-PV (E2 and E3, respectively). *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (H) Western blot analysis of WT and FBXO11 −/− macrophages treated with LukS-PV over time. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages at respective time points. Recombinant LukS-PV is included in the left lane. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001;by two-way ANOVA with Sidak’s multiple comparisons test. Source data are available for this figure.
    Figure Legend Snippet: (A) Live cell imaging showing the percentage of Draq7-positive (dead) WT and C5aR1 −/− macrophages to PBS or PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. ** = P < 0.01 for WT versus C5aR1 −/− at 15 h post toxin treatment; by unpaired t test. (B) Flow cytometric analysis and median fluorescence intensity of C5aR1 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, red and blue lines represent FBXO11 −/− (E2 and E3, respectively), and dark grey line represents C5aR1 −/− macrophages. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Confocal microscopy images of WT, FBXO11 −/− , and C5aR1 −/− macrophages. PFA-fixed cells were stained using anti-C5aR1 (green), and nuclei were stained with DAPI (blue). Scale bar corresponds to 100 μM. (D) Immunoblot analysis of C5aR1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of C5aR1 in WT macrophages that were treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophage. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (F) qRT-PCR analysis of C5aR1 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. C5aR1 levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (G) Flow cytometric analysis and MFI of recombinant LukS-PV subunit binding to WT and FBXO11 −/− macrophages. Grey line represents isotype, dotted black line represents no LukS-PV negative control, solid black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages treated with LukS-PV (E2 and E3, respectively). *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (H) Western blot analysis of WT and FBXO11 −/− macrophages treated with LukS-PV over time. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages at respective time points. Recombinant LukS-PV is included in the left lane. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001;by two-way ANOVA with Sidak’s multiple comparisons test. Source data are available for this figure.

    Techniques Used: Live Cell Imaging, Fluorescence, Expressing, Negative Control, Confocal Microscopy, Staining, Western Blot, Quantitative RT-PCR, Recombinant, Binding Assay

    (A) Western blot analysis of C5aR1 and FBXO11 protein expression in WT and FBXO11 −/− (E2) macrophages, exposed to LPS (100 ng/ml), S. aureus (MOI 10), or heat-killed S. aureus (MOI 10) for 3 h. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (B, C) Flow cytometric analysis and MFI of (B) CD45 and (C) CD11b protein expression in untreated (black line) and LPS-treated (red line) WT, and FBXO11 −/− macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated WT and FBXO11 −/− macrophages treated with LukAB (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for +LukAB versus + LukAB +LPS at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
    Figure Legend Snippet: (A) Western blot analysis of C5aR1 and FBXO11 protein expression in WT and FBXO11 −/− (E2) macrophages, exposed to LPS (100 ng/ml), S. aureus (MOI 10), or heat-killed S. aureus (MOI 10) for 3 h. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (B, C) Flow cytometric analysis and MFI of (B) CD45 and (C) CD11b protein expression in untreated (black line) and LPS-treated (red line) WT, and FBXO11 −/− macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated WT and FBXO11 −/− macrophages treated with LukAB (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for +LukAB versus + LukAB +LPS at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Techniques Used: Western Blot, Expressing, Live Cell Imaging

    (A, B) WT, FBXO11 −/− , and (B) C5aR1 −/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11 −/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11 −/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11 −/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11 −/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are available for this figure.
    Figure Legend Snippet: (A, B) WT, FBXO11 −/− , and (B) C5aR1 −/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11 −/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11 −/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11 −/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11 −/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are available for this figure.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Isolation, Quantitative RT-PCR

    (A) Western blot analysis of BCL-6 and CD40 in WT and FBXO11 −/− macrophage whole-cell lysate. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (B) qRT-PCR analysis of CD40 mRNA in WT and FBXO11 −/− macrophages. CD40 mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Flow cytometric analysis and MFI of CD40 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Western blot analysis of BCL-6, CD40, C5aR1, and IL-1β in WT and FBXO11 −/− macrophage whole-cell lysate treated with or without BI-3802 (1 or 5 μM) during recovery period. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages at respective time points. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR analysis of CD40, C5aR1, and IL-1β mRNA in WT and FBXO11 −/− macrophages treated with or without BI-3802 (5 μM). mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (F) BI-3802 (5 μM) untreated or treated WT, FBXO11 −/− (E2) macrophages were treated with PVL (62.5 ng/ml), LukAB (62.5 ng/ml) or nigericin (10 μM) for 2 h. Il-1β levels in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. Source data are available for this figure.
    Figure Legend Snippet: (A) Western blot analysis of BCL-6 and CD40 in WT and FBXO11 −/− macrophage whole-cell lysate. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (B) qRT-PCR analysis of CD40 mRNA in WT and FBXO11 −/− macrophages. CD40 mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Flow cytometric analysis and MFI of CD40 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Western blot analysis of BCL-6, CD40, C5aR1, and IL-1β in WT and FBXO11 −/− macrophage whole-cell lysate treated with or without BI-3802 (1 or 5 μM) during recovery period. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages at respective time points. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR analysis of CD40, C5aR1, and IL-1β mRNA in WT and FBXO11 −/− macrophages treated with or without BI-3802 (5 μM). mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (F) BI-3802 (5 μM) untreated or treated WT, FBXO11 −/− (E2) macrophages were treated with PVL (62.5 ng/ml), LukAB (62.5 ng/ml) or nigericin (10 μM) for 2 h. Il-1β levels in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. Source data are available for this figure.

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

    4970l  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4970l
    4970l, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit monoclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal antibody
    Rabbit Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti human mouse beta actin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti human mouse beta actin

    Rabbit Polyclonal Anti Human Mouse Beta Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Carbon source availability drives nutrient utilization in CD8 + T cells"

    Article Title: Carbon source availability drives nutrient utilization in CD8 + T cells

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2022.07.012


    Figure Legend Snippet:

    Techniques Used: Luciferase, shRNA, Plasmid Preparation, Recombinant, Staining, Cell Isolation, Selection, Protease Inhibitor, Software

    beta actin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc beta actin
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    rabbit anti actb polyclonal igg  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti actb polyclonal igg
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    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised <t>to</t> <t>α-tubulin</t> and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
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    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised <t>to</t> <t>α-tubulin</t> and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
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    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised <t>to</t> <t>α-tubulin</t> and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.
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    (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    doi: 10.26508/lsa.202201735

    Figure Lengend Snippet: (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Article Snippet: Anti-β-actin (#8457; CST) or anti-α-tubulin (#3873; CST) was used as a loading control.

    Techniques: Genome Wide, CRISPR, DNA Sequencing, Sequencing, Quantitative RT-PCR, Western Blot, Microscopy, Live Cell Imaging

    (A) Live cell imaging showing the percentage of Draq7-positive (dead) WT and C5aR1 −/− macrophages to PBS or PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. ** = P < 0.01 for WT versus C5aR1 −/− at 15 h post toxin treatment; by unpaired t test. (B) Flow cytometric analysis and median fluorescence intensity of C5aR1 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, red and blue lines represent FBXO11 −/− (E2 and E3, respectively), and dark grey line represents C5aR1 −/− macrophages. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Confocal microscopy images of WT, FBXO11 −/− , and C5aR1 −/− macrophages. PFA-fixed cells were stained using anti-C5aR1 (green), and nuclei were stained with DAPI (blue). Scale bar corresponds to 100 μM. (D) Immunoblot analysis of C5aR1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of C5aR1 in WT macrophages that were treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophage. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (F) qRT-PCR analysis of C5aR1 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. C5aR1 levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (G) Flow cytometric analysis and MFI of recombinant LukS-PV subunit binding to WT and FBXO11 −/− macrophages. Grey line represents isotype, dotted black line represents no LukS-PV negative control, solid black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages treated with LukS-PV (E2 and E3, respectively). *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (H) Western blot analysis of WT and FBXO11 −/− macrophages treated with LukS-PV over time. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages at respective time points. Recombinant LukS-PV is included in the left lane. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001;by two-way ANOVA with Sidak’s multiple comparisons test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    doi: 10.26508/lsa.202201735

    Figure Lengend Snippet: (A) Live cell imaging showing the percentage of Draq7-positive (dead) WT and C5aR1 −/− macrophages to PBS or PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. ** = P < 0.01 for WT versus C5aR1 −/− at 15 h post toxin treatment; by unpaired t test. (B) Flow cytometric analysis and median fluorescence intensity of C5aR1 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, red and blue lines represent FBXO11 −/− (E2 and E3, respectively), and dark grey line represents C5aR1 −/− macrophages. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Confocal microscopy images of WT, FBXO11 −/− , and C5aR1 −/− macrophages. PFA-fixed cells were stained using anti-C5aR1 (green), and nuclei were stained with DAPI (blue). Scale bar corresponds to 100 μM. (D) Immunoblot analysis of C5aR1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of C5aR1 in WT macrophages that were treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophage. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (F) qRT-PCR analysis of C5aR1 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. C5aR1 levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (G) Flow cytometric analysis and MFI of recombinant LukS-PV subunit binding to WT and FBXO11 −/− macrophages. Grey line represents isotype, dotted black line represents no LukS-PV negative control, solid black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages treated with LukS-PV (E2 and E3, respectively). *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (H) Western blot analysis of WT and FBXO11 −/− macrophages treated with LukS-PV over time. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages at respective time points. Recombinant LukS-PV is included in the left lane. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001;by two-way ANOVA with Sidak’s multiple comparisons test. Source data are available for this figure.

    Article Snippet: Anti-β-actin (#8457; CST) or anti-α-tubulin (#3873; CST) was used as a loading control.

    Techniques: Live Cell Imaging, Fluorescence, Expressing, Negative Control, Confocal Microscopy, Staining, Western Blot, Quantitative RT-PCR, Recombinant, Binding Assay

    (A) Western blot analysis of C5aR1 and FBXO11 protein expression in WT and FBXO11 −/− (E2) macrophages, exposed to LPS (100 ng/ml), S. aureus (MOI 10), or heat-killed S. aureus (MOI 10) for 3 h. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (B, C) Flow cytometric analysis and MFI of (B) CD45 and (C) CD11b protein expression in untreated (black line) and LPS-treated (red line) WT, and FBXO11 −/− macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated WT and FBXO11 −/− macrophages treated with LukAB (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for +LukAB versus + LukAB +LPS at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    doi: 10.26508/lsa.202201735

    Figure Lengend Snippet: (A) Western blot analysis of C5aR1 and FBXO11 protein expression in WT and FBXO11 −/− (E2) macrophages, exposed to LPS (100 ng/ml), S. aureus (MOI 10), or heat-killed S. aureus (MOI 10) for 3 h. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (B, C) Flow cytometric analysis and MFI of (B) CD45 and (C) CD11b protein expression in untreated (black line) and LPS-treated (red line) WT, and FBXO11 −/− macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated WT and FBXO11 −/− macrophages treated with LukAB (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for +LukAB versus + LukAB +LPS at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

    Article Snippet: Anti-β-actin (#8457; CST) or anti-α-tubulin (#3873; CST) was used as a loading control.

    Techniques: Western Blot, Expressing, Live Cell Imaging

    (A, B) WT, FBXO11 −/− , and (B) C5aR1 −/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11 −/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11 −/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11 −/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11 −/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    doi: 10.26508/lsa.202201735

    Figure Lengend Snippet: (A, B) WT, FBXO11 −/− , and (B) C5aR1 −/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11 −/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11 −/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11 −/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11 −/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are available for this figure.

    Article Snippet: Anti-β-actin (#8457; CST) or anti-α-tubulin (#3873; CST) was used as a loading control.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Isolation, Quantitative RT-PCR

    (A) Western blot analysis of BCL-6 and CD40 in WT and FBXO11 −/− macrophage whole-cell lysate. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (B) qRT-PCR analysis of CD40 mRNA in WT and FBXO11 −/− macrophages. CD40 mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Flow cytometric analysis and MFI of CD40 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Western blot analysis of BCL-6, CD40, C5aR1, and IL-1β in WT and FBXO11 −/− macrophage whole-cell lysate treated with or without BI-3802 (1 or 5 μM) during recovery period. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages at respective time points. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR analysis of CD40, C5aR1, and IL-1β mRNA in WT and FBXO11 −/− macrophages treated with or without BI-3802 (5 μM). mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (F) BI-3802 (5 μM) untreated or treated WT, FBXO11 −/− (E2) macrophages were treated with PVL (62.5 ng/ml), LukAB (62.5 ng/ml) or nigericin (10 μM) for 2 h. Il-1β levels in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. Source data are available for this figure.

    Journal: Life Science Alliance

    Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

    doi: 10.26508/lsa.202201735

    Figure Lengend Snippet: (A) Western blot analysis of BCL-6 and CD40 in WT and FBXO11 −/− macrophage whole-cell lysate. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (B) qRT-PCR analysis of CD40 mRNA in WT and FBXO11 −/− macrophages. CD40 mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Flow cytometric analysis and MFI of CD40 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Western blot analysis of BCL-6, CD40, C5aR1, and IL-1β in WT and FBXO11 −/− macrophage whole-cell lysate treated with or without BI-3802 (1 or 5 μM) during recovery period. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages at respective time points. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR analysis of CD40, C5aR1, and IL-1β mRNA in WT and FBXO11 −/− macrophages treated with or without BI-3802 (5 μM). mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (F) BI-3802 (5 μM) untreated or treated WT, FBXO11 −/− (E2) macrophages were treated with PVL (62.5 ng/ml), LukAB (62.5 ng/ml) or nigericin (10 μM) for 2 h. Il-1β levels in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. Source data are available for this figure.

    Article Snippet: Anti-β-actin (#8457; CST) or anti-α-tubulin (#3873; CST) was used as a loading control.

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

    Journal: Cell metabolism

    Article Title: Carbon source availability drives nutrient utilization in CD8 + T cells

    doi: 10.1016/j.cmet.2022.07.012

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-human/mouse beta-actin , Cell Signaling , #4967.

    Techniques: Luciferase, shRNA, Plasmid Preparation, Recombinant, Staining, Cell Isolation, Selection, Protease Inhibitor, Software