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hsp70  (R&D Systems)


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    R&D Systems hsp70
    RFA vigorously stimulates <t>HSP70</t> synthesis in DCs. C57BL/6 mice were subjected to RFA treatment. Single-cell suspensions were prepared at different timepoints followed by staining with fluorescence-conjugated viability dye, anti-CD11c, and anti-F4/80 antibodies. Cells were then permeabilized followed by intracellular staining with fluorescence-conjugated anti-HSP70 antibodies. (A) Representative dot plots showing dynamic increase of percentage of HSP70+ cells in DCs, macrophages, and non-immune cells in the first 6 h after RFA treatment or non-treated skin (baseline). (B) Percentage of HSP70+ DCs, macrophages, and non-immune cells at different timepoints till 24 h n=4. Data are representative of two independent experiments with similar results.
    Hsp70, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hsp70/product/R&D Systems
    Average 92 stars, based on 17 article reviews
    hsp70 - by Bioz Stars, 2026-05
    92/100 stars

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    1) Product Images from "Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation"

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2025.1638948

    RFA vigorously stimulates HSP70 synthesis in DCs. C57BL/6 mice were subjected to RFA treatment. Single-cell suspensions were prepared at different timepoints followed by staining with fluorescence-conjugated viability dye, anti-CD11c, and anti-F4/80 antibodies. Cells were then permeabilized followed by intracellular staining with fluorescence-conjugated anti-HSP70 antibodies. (A) Representative dot plots showing dynamic increase of percentage of HSP70+ cells in DCs, macrophages, and non-immune cells in the first 6 h after RFA treatment or non-treated skin (baseline). (B) Percentage of HSP70+ DCs, macrophages, and non-immune cells at different timepoints till 24 h n=4. Data are representative of two independent experiments with similar results.
    Figure Legend Snippet: RFA vigorously stimulates HSP70 synthesis in DCs. C57BL/6 mice were subjected to RFA treatment. Single-cell suspensions were prepared at different timepoints followed by staining with fluorescence-conjugated viability dye, anti-CD11c, and anti-F4/80 antibodies. Cells were then permeabilized followed by intracellular staining with fluorescence-conjugated anti-HSP70 antibodies. (A) Representative dot plots showing dynamic increase of percentage of HSP70+ cells in DCs, macrophages, and non-immune cells in the first 6 h after RFA treatment or non-treated skin (baseline). (B) Percentage of HSP70+ DCs, macrophages, and non-immune cells at different timepoints till 24 h n=4. Data are representative of two independent experiments with similar results.

    Techniques Used: Staining, Fluorescence

    Separation of RFA-induced HSP70 from constitutively expressed HSc70. (A) Lateral back skin of WT and HSP70 KO mice were subjected to RFA or Sham treatment. RFA and Sham-treated skin (1×1 cm 2 ) was collected 18 h later, homogenized, and purified with ADP-Agarose column. Eluted samples were concentrated and adjusted to the same volume of 100 µl. The same volume (10 µl) of WT samples isolated from 4 pieces of skin, KO samples isolated from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and silver staining. (B) WT (10 µl) and KO samples (30 µl), both purified from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and western blotting analysis with anti-HSP70 antibodies (upper) or anti-HSc70 antibodies (lower). The volume of KO samples was increased for readiness detection of HSP70 if there was any. 1. Recombinant mouse HSP70; 2. Purified sample from RFA-treated WT skin; 3. Purified sample from Sham-treated WT skin; 4. Purified sample from RFA-treated HSP70 KO skin; 5. Purified sample from Sham-treated HSP70 KO skin. Full membrane pictures were shown in <xref ref-type= Supplementary Figure S2 . (C, D) Densitometry analysis of relative HSP70 (C) and HSc70 levels (D) of Figure 2B in WT and HSP70 KO mice using ImageJ. Results were the combination of two independent experiments with similar results. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare differences between Sham and RFA groups. N.D., not detectable. NS, not significant. ***, p<0.001. " title="Separation of RFA-induced HSP70 from constitutively expressed HSc70. (A) Lateral back skin ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Separation of RFA-induced HSP70 from constitutively expressed HSc70. (A) Lateral back skin of WT and HSP70 KO mice were subjected to RFA or Sham treatment. RFA and Sham-treated skin (1×1 cm 2 ) was collected 18 h later, homogenized, and purified with ADP-Agarose column. Eluted samples were concentrated and adjusted to the same volume of 100 µl. The same volume (10 µl) of WT samples isolated from 4 pieces of skin, KO samples isolated from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and silver staining. (B) WT (10 µl) and KO samples (30 µl), both purified from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and western blotting analysis with anti-HSP70 antibodies (upper) or anti-HSc70 antibodies (lower). The volume of KO samples was increased for readiness detection of HSP70 if there was any. 1. Recombinant mouse HSP70; 2. Purified sample from RFA-treated WT skin; 3. Purified sample from Sham-treated WT skin; 4. Purified sample from RFA-treated HSP70 KO skin; 5. Purified sample from Sham-treated HSP70 KO skin. Full membrane pictures were shown in Supplementary Figure S2 . (C, D) Densitometry analysis of relative HSP70 (C) and HSc70 levels (D) of Figure 2B in WT and HSP70 KO mice using ImageJ. Results were the combination of two independent experiments with similar results. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare differences between Sham and RFA groups. N.D., not detectable. NS, not significant. ***, p<0.001.

    Techniques Used: Purification, Isolation, Recombinant, SDS Page, Silver Staining, Western Blot, Membrane

    RFA stimulates HSP70 synthesis and extracellular release. (A, B) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was dissected right after treatment and further cut into 1 mm wide slices for culture in 24-well plates. A small volume (100 µl) of medium was added to just cover the tissue. Culture medium was harvested 18 h later to measure HSP70 levels (A) . Skin was also harvested at the same time and homogenized in T-PER buffer for measurement of HSP70 levels (B) . (C, D) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was then subjected to one pulse of AFL treatment at 5mJ energy and 10% coverage. Powder mannitol-coated reservoir patches were then topically applied to extract interstitial fluid via skin microchannels. Powder reservoir patches (C) and underneath skin (D) were harvested 24 h later for measurement of HSP70 levels. n=5 in A, B and n=6 in (C, D) Two-tailed student’s t-test was used to compare differences between groups. *, p<0.05; **, p<0.01. Data are representative of two independent experiments with similar results.
    Figure Legend Snippet: RFA stimulates HSP70 synthesis and extracellular release. (A, B) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was dissected right after treatment and further cut into 1 mm wide slices for culture in 24-well plates. A small volume (100 µl) of medium was added to just cover the tissue. Culture medium was harvested 18 h later to measure HSP70 levels (A) . Skin was also harvested at the same time and homogenized in T-PER buffer for measurement of HSP70 levels (B) . (C, D) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was then subjected to one pulse of AFL treatment at 5mJ energy and 10% coverage. Powder mannitol-coated reservoir patches were then topically applied to extract interstitial fluid via skin microchannels. Powder reservoir patches (C) and underneath skin (D) were harvested 24 h later for measurement of HSP70 levels. n=5 in A, B and n=6 in (C, D) Two-tailed student’s t-test was used to compare differences between groups. *, p<0.05; **, p<0.01. Data are representative of two independent experiments with similar results.

    Techniques Used: Two Tailed Test

    Evidence of in situ HSP70/OVA association. Lateral back skin of WT or HSP70 KO mice was subjected to RFA or Sham treatment followed by ID injection of 1 µg OVA into RFA or Sham-treated skin. Skin was collected 6 h later. (A–C) Skin was subjected to cryo-sectioning and PLA analysis of HSP70/OVA binding. Representative HSP70/OVA PLA results in WT mice were shown in A . Quantitative analysis of HSP70/OVA PLA signals in randomly selected regions of 1.6 mm 2 in WT mice and HSP70 KO mice was shown in (B, C) , respectively. (D, E) Lateral back skin of WT and HSP70 KO mice (n=2) were exposed to RFA (left) or Sham treatment (right) followed by ID OVA injection. Skin was collected 6 h later and homogenized in RIPA buffer. The same amounts of total proteins were incubated with anti-OVA antibodies and then protein A/G agarose followed by centrifugation and washing. After boiling, IP samples and tissue lysates were subjected to SDS-PAGE and IB detection of HSP70 and OVA in WT (D) and HSP70 KO mice (E) . Intact membrane pictures were shown in <xref ref-type= Supplementary Figure S4 . Arrows in A point to positive PLA signals. Scale in A : 100 µm. Two-tailed student’s t-test was used to compare differences between groups in (B, C) . n=31–33 in B and n=7 in C . ***, p<0.001. NS, not significant. Data are representative of two independent experiments with similar results. " title="Evidence of in situ HSP70/OVA association. Lateral back skin of WT or HSP70 ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Evidence of in situ HSP70/OVA association. Lateral back skin of WT or HSP70 KO mice was subjected to RFA or Sham treatment followed by ID injection of 1 µg OVA into RFA or Sham-treated skin. Skin was collected 6 h later. (A–C) Skin was subjected to cryo-sectioning and PLA analysis of HSP70/OVA binding. Representative HSP70/OVA PLA results in WT mice were shown in A . Quantitative analysis of HSP70/OVA PLA signals in randomly selected regions of 1.6 mm 2 in WT mice and HSP70 KO mice was shown in (B, C) , respectively. (D, E) Lateral back skin of WT and HSP70 KO mice (n=2) were exposed to RFA (left) or Sham treatment (right) followed by ID OVA injection. Skin was collected 6 h later and homogenized in RIPA buffer. The same amounts of total proteins were incubated with anti-OVA antibodies and then protein A/G agarose followed by centrifugation and washing. After boiling, IP samples and tissue lysates were subjected to SDS-PAGE and IB detection of HSP70 and OVA in WT (D) and HSP70 KO mice (E) . Intact membrane pictures were shown in Supplementary Figure S4 . Arrows in A point to positive PLA signals. Scale in A : 100 µm. Two-tailed student’s t-test was used to compare differences between groups in (B, C) . n=31–33 in B and n=7 in C . ***, p<0.001. NS, not significant. Data are representative of two independent experiments with similar results.

    Techniques Used: In Situ, Injection, Binding Assay, Incubation, Centrifugation, SDS Page, Membrane, Two Tailed Test

    HSP70 contributes to RFA-enhanced antigen uptake in skin and draining LNs. WT and HSP70 KO were subjected to RFA or Sham treatment followed by ID injection of 2 µg AF647-OVA into RF or Sham-treated skin. Skin and draining LNs were dissected 20 h later followed by single-cell suspension preparation, immunostaining, and flow cytometry analysis. Gating strategies were shown in <xref ref-type= Supplementary Figures S1B, C . (A) Percentages of AF647-OVA+ cells in skin DCs (CD11c+ MHC II+) in WT and HSP70 KO mice. (B) Representative histogram of AF647-OVA levels in skin DCs of different groups in WT and HSP70 KO mice. (C) Representative dot plots of AF647-OVA+ migDCs of different groups in WT and HSP70 KO mice. (D) Percentage of AF647-OVA+ cells in migDC (D) , cDC (E) , and pDC (F) in WT and HSP70 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=8. *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results. " title="HSP70 contributes to RFA-enhanced antigen uptake in skin and ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: HSP70 contributes to RFA-enhanced antigen uptake in skin and draining LNs. WT and HSP70 KO were subjected to RFA or Sham treatment followed by ID injection of 2 µg AF647-OVA into RF or Sham-treated skin. Skin and draining LNs were dissected 20 h later followed by single-cell suspension preparation, immunostaining, and flow cytometry analysis. Gating strategies were shown in Supplementary Figures S1B, C . (A) Percentages of AF647-OVA+ cells in skin DCs (CD11c+ MHC II+) in WT and HSP70 KO mice. (B) Representative histogram of AF647-OVA levels in skin DCs of different groups in WT and HSP70 KO mice. (C) Representative dot plots of AF647-OVA+ migDCs of different groups in WT and HSP70 KO mice. (D) Percentage of AF647-OVA+ cells in migDC (D) , cDC (E) , and pDC (F) in WT and HSP70 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=8. *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results.

    Techniques Used: Injection, Suspension, Immunostaining, Flow Cytometry

    RFA-induced HSP70 lacks the ability to induce DC maturation. (A) The same skin cell samples in <xref ref-type= Figures 5A, B were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in CD11c+ DCs of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (B–D) . The same LN samples as in Figures 5C, D were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in cDC (B) , migDC (C) , and pDC (D) of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (E) BMDCs were incubated with AF647-OVA alone (control) or in the presence of HSc70 or HSP70-rich elutes or LPS. Cells were analyzed for their surface expression of CD40, CD80, and CD86 24 h later. MFI of CD40, CD80, and CD86 was compared between groups. Two-way ANOVA with Newman-Keuls’s multiple comparison test was used to compare differences between groups in A-D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences between groups in (E) n=6–8 in A-D. n=6 in (E) *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results. " title="RFA-induced HSP70 lacks the ability to induce DC maturation. (A) ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: RFA-induced HSP70 lacks the ability to induce DC maturation. (A) The same skin cell samples in Figures 5A, B were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in CD11c+ DCs of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (B–D) . The same LN samples as in Figures 5C, D were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in cDC (B) , migDC (C) , and pDC (D) of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (E) BMDCs were incubated with AF647-OVA alone (control) or in the presence of HSc70 or HSP70-rich elutes or LPS. Cells were analyzed for their surface expression of CD40, CD80, and CD86 24 h later. MFI of CD40, CD80, and CD86 was compared between groups. Two-way ANOVA with Newman-Keuls’s multiple comparison test was used to compare differences between groups in A-D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences between groups in (E) n=6–8 in A-D. n=6 in (E) *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results.

    Techniques Used: Expressing, Incubation, Control, Comparison

    HSP70 suppresses RFA-induced TLR4/IRAK/NFκB signaling. (A, B) WT and HSP70 KO mice were subjected to RFA or Sham treatment or ID injection of LPS or PBS. Skin was collected 6 h later in (A, B) IP and IB were conducted to evaluate TLR4/TIRAP binding (A) and IRAK4/IRAK1 binding in (B, C) . WT, HSP70 KO, MyD88 KO, TLR2 KO, and TLR4 KO mice were subjected to RFA or Sham treatment or ID injection of LPS. Skin was collected 2 h later. Cytosol and nuclear fractions were separated and nuclear fraction was analyzed by WB analysis to detect phosphorylated p65 using Lamin b1 as a loading control. (D) Skin IL-6 levels 6 h after RFA, Sham, or LPS treatment of lateral back skin of WT, HSP70 KO, TLR2 KO, TLR4 KO, and MyD88 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=4-6. *, p<0.05; **, p<0.01; ***, p<0.001. Original membrane pictures were shown in <xref ref-type= Supplementary Figure S9 . Data are representative of two independent experiments with similar results. " title="HSP70 suppresses RFA-induced TLR4/IRAK/NFκB signaling. (A, B) WT and ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: HSP70 suppresses RFA-induced TLR4/IRAK/NFκB signaling. (A, B) WT and HSP70 KO mice were subjected to RFA or Sham treatment or ID injection of LPS or PBS. Skin was collected 6 h later in (A, B) IP and IB were conducted to evaluate TLR4/TIRAP binding (A) and IRAK4/IRAK1 binding in (B, C) . WT, HSP70 KO, MyD88 KO, TLR2 KO, and TLR4 KO mice were subjected to RFA or Sham treatment or ID injection of LPS. Skin was collected 2 h later. Cytosol and nuclear fractions were separated and nuclear fraction was analyzed by WB analysis to detect phosphorylated p65 using Lamin b1 as a loading control. (D) Skin IL-6 levels 6 h after RFA, Sham, or LPS treatment of lateral back skin of WT, HSP70 KO, TLR2 KO, TLR4 KO, and MyD88 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=4-6. *, p<0.05; **, p<0.01; ***, p<0.001. Original membrane pictures were shown in Supplementary Figure S9 . Data are representative of two independent experiments with similar results.

    Techniques Used: Injection, Binding Assay, Control, Membrane



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    Image Search Results


    RFA vigorously stimulates HSP70 synthesis in DCs. C57BL/6 mice were subjected to RFA treatment. Single-cell suspensions were prepared at different timepoints followed by staining with fluorescence-conjugated viability dye, anti-CD11c, and anti-F4/80 antibodies. Cells were then permeabilized followed by intracellular staining with fluorescence-conjugated anti-HSP70 antibodies. (A) Representative dot plots showing dynamic increase of percentage of HSP70+ cells in DCs, macrophages, and non-immune cells in the first 6 h after RFA treatment or non-treated skin (baseline). (B) Percentage of HSP70+ DCs, macrophages, and non-immune cells at different timepoints till 24 h n=4. Data are representative of two independent experiments with similar results.

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: RFA vigorously stimulates HSP70 synthesis in DCs. C57BL/6 mice were subjected to RFA treatment. Single-cell suspensions were prepared at different timepoints followed by staining with fluorescence-conjugated viability dye, anti-CD11c, and anti-F4/80 antibodies. Cells were then permeabilized followed by intracellular staining with fluorescence-conjugated anti-HSP70 antibodies. (A) Representative dot plots showing dynamic increase of percentage of HSP70+ cells in DCs, macrophages, and non-immune cells in the first 6 h after RFA treatment or non-treated skin (baseline). (B) Percentage of HSP70+ DCs, macrophages, and non-immune cells at different timepoints till 24 h n=4. Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Staining, Fluorescence

    Separation of RFA-induced HSP70 from constitutively expressed HSc70. (A) Lateral back skin of WT and HSP70 KO mice were subjected to RFA or Sham treatment. RFA and Sham-treated skin (1×1 cm 2 ) was collected 18 h later, homogenized, and purified with ADP-Agarose column. Eluted samples were concentrated and adjusted to the same volume of 100 µl. The same volume (10 µl) of WT samples isolated from 4 pieces of skin, KO samples isolated from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and silver staining. (B) WT (10 µl) and KO samples (30 µl), both purified from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and western blotting analysis with anti-HSP70 antibodies (upper) or anti-HSc70 antibodies (lower). The volume of KO samples was increased for readiness detection of HSP70 if there was any. 1. Recombinant mouse HSP70; 2. Purified sample from RFA-treated WT skin; 3. Purified sample from Sham-treated WT skin; 4. Purified sample from RFA-treated HSP70 KO skin; 5. Purified sample from Sham-treated HSP70 KO skin. Full membrane pictures were shown in <xref ref-type= Supplementary Figure S2 . (C, D) Densitometry analysis of relative HSP70 (C) and HSc70 levels (D) of Figure 2B in WT and HSP70 KO mice using ImageJ. Results were the combination of two independent experiments with similar results. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare differences between Sham and RFA groups. N.D., not detectable. NS, not significant. ***, p<0.001. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: Separation of RFA-induced HSP70 from constitutively expressed HSc70. (A) Lateral back skin of WT and HSP70 KO mice were subjected to RFA or Sham treatment. RFA and Sham-treated skin (1×1 cm 2 ) was collected 18 h later, homogenized, and purified with ADP-Agarose column. Eluted samples were concentrated and adjusted to the same volume of 100 µl. The same volume (10 µl) of WT samples isolated from 4 pieces of skin, KO samples isolated from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and silver staining. (B) WT (10 µl) and KO samples (30 µl), both purified from one piece of skin, and recombinant murine HSP70 were subjected to SDS-PAGE separation and western blotting analysis with anti-HSP70 antibodies (upper) or anti-HSc70 antibodies (lower). The volume of KO samples was increased for readiness detection of HSP70 if there was any. 1. Recombinant mouse HSP70; 2. Purified sample from RFA-treated WT skin; 3. Purified sample from Sham-treated WT skin; 4. Purified sample from RFA-treated HSP70 KO skin; 5. Purified sample from Sham-treated HSP70 KO skin. Full membrane pictures were shown in Supplementary Figure S2 . (C, D) Densitometry analysis of relative HSP70 (C) and HSc70 levels (D) of Figure 2B in WT and HSP70 KO mice using ImageJ. Results were the combination of two independent experiments with similar results. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare differences between Sham and RFA groups. N.D., not detectable. NS, not significant. ***, p<0.001.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Purification, Isolation, Recombinant, SDS Page, Silver Staining, Western Blot, Membrane

    RFA stimulates HSP70 synthesis and extracellular release. (A, B) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was dissected right after treatment and further cut into 1 mm wide slices for culture in 24-well plates. A small volume (100 µl) of medium was added to just cover the tissue. Culture medium was harvested 18 h later to measure HSP70 levels (A) . Skin was also harvested at the same time and homogenized in T-PER buffer for measurement of HSP70 levels (B) . (C, D) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was then subjected to one pulse of AFL treatment at 5mJ energy and 10% coverage. Powder mannitol-coated reservoir patches were then topically applied to extract interstitial fluid via skin microchannels. Powder reservoir patches (C) and underneath skin (D) were harvested 24 h later for measurement of HSP70 levels. n=5 in A, B and n=6 in (C, D) Two-tailed student’s t-test was used to compare differences between groups. *, p<0.05; **, p<0.01. Data are representative of two independent experiments with similar results.

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: RFA stimulates HSP70 synthesis and extracellular release. (A, B) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was dissected right after treatment and further cut into 1 mm wide slices for culture in 24-well plates. A small volume (100 µl) of medium was added to just cover the tissue. Culture medium was harvested 18 h later to measure HSP70 levels (A) . Skin was also harvested at the same time and homogenized in T-PER buffer for measurement of HSP70 levels (B) . (C, D) Lateral back skin (1.2×1.2 cm 2 ) of C57BL/6 mice was subjected to RFA or Sham treatment. Skin was then subjected to one pulse of AFL treatment at 5mJ energy and 10% coverage. Powder mannitol-coated reservoir patches were then topically applied to extract interstitial fluid via skin microchannels. Powder reservoir patches (C) and underneath skin (D) were harvested 24 h later for measurement of HSP70 levels. n=5 in A, B and n=6 in (C, D) Two-tailed student’s t-test was used to compare differences between groups. *, p<0.05; **, p<0.01. Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Two Tailed Test

    Evidence of in situ HSP70/OVA association. Lateral back skin of WT or HSP70 KO mice was subjected to RFA or Sham treatment followed by ID injection of 1 µg OVA into RFA or Sham-treated skin. Skin was collected 6 h later. (A–C) Skin was subjected to cryo-sectioning and PLA analysis of HSP70/OVA binding. Representative HSP70/OVA PLA results in WT mice were shown in A . Quantitative analysis of HSP70/OVA PLA signals in randomly selected regions of 1.6 mm 2 in WT mice and HSP70 KO mice was shown in (B, C) , respectively. (D, E) Lateral back skin of WT and HSP70 KO mice (n=2) were exposed to RFA (left) or Sham treatment (right) followed by ID OVA injection. Skin was collected 6 h later and homogenized in RIPA buffer. The same amounts of total proteins were incubated with anti-OVA antibodies and then protein A/G agarose followed by centrifugation and washing. After boiling, IP samples and tissue lysates were subjected to SDS-PAGE and IB detection of HSP70 and OVA in WT (D) and HSP70 KO mice (E) . Intact membrane pictures were shown in <xref ref-type= Supplementary Figure S4 . Arrows in A point to positive PLA signals. Scale in A : 100 µm. Two-tailed student’s t-test was used to compare differences between groups in (B, C) . n=31–33 in B and n=7 in C . ***, p<0.001. NS, not significant. Data are representative of two independent experiments with similar results. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: Evidence of in situ HSP70/OVA association. Lateral back skin of WT or HSP70 KO mice was subjected to RFA or Sham treatment followed by ID injection of 1 µg OVA into RFA or Sham-treated skin. Skin was collected 6 h later. (A–C) Skin was subjected to cryo-sectioning and PLA analysis of HSP70/OVA binding. Representative HSP70/OVA PLA results in WT mice were shown in A . Quantitative analysis of HSP70/OVA PLA signals in randomly selected regions of 1.6 mm 2 in WT mice and HSP70 KO mice was shown in (B, C) , respectively. (D, E) Lateral back skin of WT and HSP70 KO mice (n=2) were exposed to RFA (left) or Sham treatment (right) followed by ID OVA injection. Skin was collected 6 h later and homogenized in RIPA buffer. The same amounts of total proteins were incubated with anti-OVA antibodies and then protein A/G agarose followed by centrifugation and washing. After boiling, IP samples and tissue lysates were subjected to SDS-PAGE and IB detection of HSP70 and OVA in WT (D) and HSP70 KO mice (E) . Intact membrane pictures were shown in Supplementary Figure S4 . Arrows in A point to positive PLA signals. Scale in A : 100 µm. Two-tailed student’s t-test was used to compare differences between groups in (B, C) . n=31–33 in B and n=7 in C . ***, p<0.001. NS, not significant. Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: In Situ, Injection, Binding Assay, Incubation, Centrifugation, SDS Page, Membrane, Two Tailed Test

    HSP70 contributes to RFA-enhanced antigen uptake in skin and draining LNs. WT and HSP70 KO were subjected to RFA or Sham treatment followed by ID injection of 2 µg AF647-OVA into RF or Sham-treated skin. Skin and draining LNs were dissected 20 h later followed by single-cell suspension preparation, immunostaining, and flow cytometry analysis. Gating strategies were shown in <xref ref-type= Supplementary Figures S1B, C . (A) Percentages of AF647-OVA+ cells in skin DCs (CD11c+ MHC II+) in WT and HSP70 KO mice. (B) Representative histogram of AF647-OVA levels in skin DCs of different groups in WT and HSP70 KO mice. (C) Representative dot plots of AF647-OVA+ migDCs of different groups in WT and HSP70 KO mice. (D) Percentage of AF647-OVA+ cells in migDC (D) , cDC (E) , and pDC (F) in WT and HSP70 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=8. *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: HSP70 contributes to RFA-enhanced antigen uptake in skin and draining LNs. WT and HSP70 KO were subjected to RFA or Sham treatment followed by ID injection of 2 µg AF647-OVA into RF or Sham-treated skin. Skin and draining LNs were dissected 20 h later followed by single-cell suspension preparation, immunostaining, and flow cytometry analysis. Gating strategies were shown in Supplementary Figures S1B, C . (A) Percentages of AF647-OVA+ cells in skin DCs (CD11c+ MHC II+) in WT and HSP70 KO mice. (B) Representative histogram of AF647-OVA levels in skin DCs of different groups in WT and HSP70 KO mice. (C) Representative dot plots of AF647-OVA+ migDCs of different groups in WT and HSP70 KO mice. (D) Percentage of AF647-OVA+ cells in migDC (D) , cDC (E) , and pDC (F) in WT and HSP70 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=8. *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Injection, Suspension, Immunostaining, Flow Cytometry

    RFA-induced HSP70 lacks the ability to induce DC maturation. (A) The same skin cell samples in <xref ref-type= Figures 5A, B were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in CD11c+ DCs of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (B–D) . The same LN samples as in Figures 5C, D were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in cDC (B) , migDC (C) , and pDC (D) of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (E) BMDCs were incubated with AF647-OVA alone (control) or in the presence of HSc70 or HSP70-rich elutes or LPS. Cells were analyzed for their surface expression of CD40, CD80, and CD86 24 h later. MFI of CD40, CD80, and CD86 was compared between groups. Two-way ANOVA with Newman-Keuls’s multiple comparison test was used to compare differences between groups in A-D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences between groups in (E) n=6–8 in A-D. n=6 in (E) *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: RFA-induced HSP70 lacks the ability to induce DC maturation. (A) The same skin cell samples in Figures 5A, B were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in CD11c+ DCs of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (B–D) . The same LN samples as in Figures 5C, D were analyzed for expression of surface co-stimulatory molecules (CD40, CD80, and CD86) in cDC (B) , migDC (C) , and pDC (D) of WT and HSP70 KO mice. MFI of CD40, CD80, and CD86 was compared between groups. (E) BMDCs were incubated with AF647-OVA alone (control) or in the presence of HSc70 or HSP70-rich elutes or LPS. Cells were analyzed for their surface expression of CD40, CD80, and CD86 24 h later. MFI of CD40, CD80, and CD86 was compared between groups. Two-way ANOVA with Newman-Keuls’s multiple comparison test was used to compare differences between groups in A-D. One-way ANOVA with Tukey’s multiple comparison test was used to compare differences between groups in (E) n=6–8 in A-D. n=6 in (E) *, p<0.05; **, p<0.01; ***, p<0.001. Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Expressing, Incubation, Control, Comparison

    HSP70 suppresses RFA-induced TLR4/IRAK/NFκB signaling. (A, B) WT and HSP70 KO mice were subjected to RFA or Sham treatment or ID injection of LPS or PBS. Skin was collected 6 h later in (A, B) IP and IB were conducted to evaluate TLR4/TIRAP binding (A) and IRAK4/IRAK1 binding in (B, C) . WT, HSP70 KO, MyD88 KO, TLR2 KO, and TLR4 KO mice were subjected to RFA or Sham treatment or ID injection of LPS. Skin was collected 2 h later. Cytosol and nuclear fractions were separated and nuclear fraction was analyzed by WB analysis to detect phosphorylated p65 using Lamin b1 as a loading control. (D) Skin IL-6 levels 6 h after RFA, Sham, or LPS treatment of lateral back skin of WT, HSP70 KO, TLR2 KO, TLR4 KO, and MyD88 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=4-6. *, p<0.05; **, p<0.01; ***, p<0.001. Original membrane pictures were shown in <xref ref-type= Supplementary Figure S9 . Data are representative of two independent experiments with similar results. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: Dual roles of in situ generated HSP70 in antigen delivery and immunoregulation

    doi: 10.3389/fimmu.2025.1638948

    Figure Lengend Snippet: HSP70 suppresses RFA-induced TLR4/IRAK/NFκB signaling. (A, B) WT and HSP70 KO mice were subjected to RFA or Sham treatment or ID injection of LPS or PBS. Skin was collected 6 h later in (A, B) IP and IB were conducted to evaluate TLR4/TIRAP binding (A) and IRAK4/IRAK1 binding in (B, C) . WT, HSP70 KO, MyD88 KO, TLR2 KO, and TLR4 KO mice were subjected to RFA or Sham treatment or ID injection of LPS. Skin was collected 2 h later. Cytosol and nuclear fractions were separated and nuclear fraction was analyzed by WB analysis to detect phosphorylated p65 using Lamin b1 as a loading control. (D) Skin IL-6 levels 6 h after RFA, Sham, or LPS treatment of lateral back skin of WT, HSP70 KO, TLR2 KO, TLR4 KO, and MyD88 KO mice. Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups. n=4-6. *, p<0.05; **, p<0.01; ***, p<0.001. Original membrane pictures were shown in Supplementary Figure S9 . Data are representative of two independent experiments with similar results.

    Article Snippet: Supernatants were subjected to SDS-PAGE and IB detection of HSP70 (MAB1663, R & D Systems), TLR4 (sc-293072, Santa Cruz), and IRAK1 (Ab238, Abcam).

    Techniques: Injection, Binding Assay, Control, Membrane