ha tag  (Addgene inc)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: TMR-Lipo-Abs protects mice from CLP-induced polymicrobial sepsis. (A–C) Schematic design of mid-grade (A) or high-grade (B, C) sepsis mouse model induced by cecal ligation and punctuation (CLP) depending on the position of the cecal ligation (top). The survival of CLP mice treated with the indicated therapy was monitored for 10 days (A) or 72 h (B, C); mortality was measured for n = 10 mice per group (bottom). Statistical differences compared with the PBS or TMR-Lipo-Abs-treated mice are indicated (log-rank test). The data are representative of three independent experimental replicates with similar results. (D) Serum cytokine levels. The data shown represent at least three independent experiments ( n ≥ 3), significance was measured by Ordinary one-way ANOVA (TNF- α , ∗∗ P = 0.0010 and ∗∗∗ P = 0.0002, IL-6, ∗∗ P = 0.0057 and ∗∗∗ P = 0.0004 and IL12p40, both ∗∗∗∗ P < 0.0001). n.s = not significant. (E) IB identification and comparison of HMGB1 and PTX3 expression in spleen, lung, and liver cells of CLP mice after different treatment as a DAMP indicator for tissue damage. (F) Representative H&E staining of the lung, spleen, and liver from 3 mice per group. Scale bar, 100 μm.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Ligation, Comparison, Expressing, Staining