antibody expression antibodies Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher antibody expressing positive control vector
    Sec63 inhibits IRE1α clustering during ER stress in cells. ( A ) HEK293 IRE1α−/− cells complemented with IRE1α-HA were transfected with either <t>control,</t> Sec63, or Sec62 siRNA. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 30h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and processed for immunostaining with <t>anti-HA</t> <t>antibodies</t> for IRE1α. Scale bars are 10 μm. ( B ) Quantification of the number of cells with IRE1α clusters from the panel A. Error bar represents standard deviation (n=2). ( C ) HEK293 IRE1α−/− cells were treated with the indicated siRNAs. After 30h of transfection, the cells were harvested and analyzed by immunoblotting for the indicated antigens. ( D ) HEK293 IRE1α−/− cells <t>expressing</t> either IRE1α-HA or IRE1α-CNX-TMD-HA were induced with 5ng/ml doxycycline and treated with either Tg or Tm for the indicated time points. The cells were then processed for immunostaining as in A. ( E ) The number of cells with IRE1α clusters were quantified from the panel D. Error bar represents standard deviation (n=2). ( F ) HEK293 IRE1α−/− cells expressing IRE1α-HA were transfected with either empty <t>vector,</t> wild type (WT) Sec63 or the J-domain mutant of Sec63. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 24h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and subsequently processed for immunostaining with anti-HA antibodies for IRE1α. ( G ) Quantification of the number of cells with IRE1α clusters from the panel F. Error bar represents standard deviation (n=2) ( H ) The cells were transfected as in the panel F, but analyzed by immunoblotting for the indicated antigens.
    Antibody Expressing Positive Control Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody expressing positive control vector/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibody expressing positive control vector - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    96
    Millipore gapdh expression
    Sec63 inhibits IRE1α clustering during ER stress in cells. ( A ) HEK293 IRE1α−/− cells complemented with IRE1α-HA were transfected with either <t>control,</t> Sec63, or Sec62 siRNA. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 30h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and processed for immunostaining with <t>anti-HA</t> <t>antibodies</t> for IRE1α. Scale bars are 10 μm. ( B ) Quantification of the number of cells with IRE1α clusters from the panel A. Error bar represents standard deviation (n=2). ( C ) HEK293 IRE1α−/− cells were treated with the indicated siRNAs. After 30h of transfection, the cells were harvested and analyzed by immunoblotting for the indicated antigens. ( D ) HEK293 IRE1α−/− cells <t>expressing</t> either IRE1α-HA or IRE1α-CNX-TMD-HA were induced with 5ng/ml doxycycline and treated with either Tg or Tm for the indicated time points. The cells were then processed for immunostaining as in A. ( E ) The number of cells with IRE1α clusters were quantified from the panel D. Error bar represents standard deviation (n=2). ( F ) HEK293 IRE1α−/− cells expressing IRE1α-HA were transfected with either empty <t>vector,</t> wild type (WT) Sec63 or the J-domain mutant of Sec63. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 24h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and subsequently processed for immunostaining with anti-HA antibodies for IRE1α. ( G ) Quantification of the number of cells with IRE1α clusters from the panel F. Error bar represents standard deviation (n=2) ( H ) The cells were transfected as in the panel F, but analyzed by immunoblotting for the indicated antigens.
    Gapdh Expression, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh expression/product/Millipore
    Average 96 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    gapdh expression - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    99
    Thermo Fisher foxp3 expression
    Short-term treatment with rIL-2/DTx leads to transient T reg depletion. ( A ) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T reg (TCRβ + CD4 + CD25 + <t>Foxp3</t> + cells) were quantified by flow cytometry at the time indicated. ( B ) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry 7 d after the final dose. ( C and D ) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10 3 metacyclic L. major promastigotes. Lesional T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry ( C ), and IgG 1 antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples ( D ), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 ( A ), n = 4–6 ( B ) and n = 5–6 ( C and D ). ( A ) ANOVA P
    Foxp3 Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/foxp3 expression/product/Thermo Fisher
    Average 99 stars, based on 1274 article reviews
    Price from $9.99 to $1999.99
    foxp3 expression - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti beta actin antibody
    Short-term treatment with rIL-2/DTx leads to transient T reg depletion. ( A ) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T reg (TCRβ + CD4 + CD25 + <t>Foxp3</t> + cells) were quantified by flow cytometry at the time indicated. ( B ) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry 7 d after the final dose. ( C and D ) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10 3 metacyclic L. major promastigotes. Lesional T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry ( C ), and IgG 1 antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples ( D ), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 ( A ), n = 4–6 ( B ) and n = 5–6 ( C and D ). ( A ) ANOVA P
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti beta actin antibody/product/Millipore
    Average 99 stars, based on 33074 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti beta actin antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    85
    Thermo Fisher anti express antibody
    Short-term treatment with rIL-2/DTx leads to transient T reg depletion. ( A ) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T reg (TCRβ + CD4 + CD25 + <t>Foxp3</t> + cells) were quantified by flow cytometry at the time indicated. ( B ) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry 7 d after the final dose. ( C and D ) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10 3 metacyclic L. major promastigotes. Lesional T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry ( C ), and IgG 1 antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples ( D ), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 ( A ), n = 4–6 ( B ) and n = 5–6 ( C and D ). ( A ) ANOVA P
    Anti Express Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti express antibody/product/Thermo Fisher
    Average 85 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    anti express antibody - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    96
    Thermo Fisher cd69 monoclonal antibody
    ARL3-Specific Release Localizes LCK to the Immune Synapse (A and F) T:B cell conjugates stained with anti ARL3, LCK, or ARL13B antibodies as indicated in the presence or absence of SEE. (B) LR of ARL3, ARL13B, and LCK of conjugates from (A) and (F): ARL3 shows no localization to the synapse in the presence (LR = 0.3) or absence of SEE (n = 38). LCK shows LR = 1.9 in the presence of SEE and 0.7 in its absence (n = 42; p ≤ 0.0001). ARL13B shows LR = 2 in the presence of SEE (n = 32) and 0.6 in its absence (n = 16; p ≤ 0.0001). (C) ARL3 WT GFP and the constitutively active ARL3 Q71L GFP were nucleofected into cells and T:B cell conjugates, in the presence of SEE, were stained with anti LCK antibodies. (D) LR of LCK of conjugates from (C). ARL3 WT GFP expression had little effect on LR of LCK (n = 18) compared to cells not expressing ARL3 WT GFP (LR = 2). ARL3 Q71L GFP-expressing cells showed LR of LCK = 1 (n = 16, p = 0.0002). Differences in LR were analyzed using the Mann-Whitney U test (B and D). (E) <t>CD69</t> expression was analyzed via flow cytometry for cells stimulated with anti CD28 and CD3 antibodies and overexpressing ARL2-GFP, ARL2 Q70L GFP, ARL3 Q71L GFP, ARL3-GFP, or GFP. ARL3 Q71L GFP (p ≤ 0.0001), ARL3 WT GFP (p = 0.004), ARL2 WT GFP (p = 0.02), and ARL2 Q70L GFP (p = 0.03) p values refer to unpaired t test data. (G) Ciliated RPE cell stained with anti ARL13B antibody. Scale bars, 5 μm.
    Cd69 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd69 monoclonal antibody/product/Thermo Fisher
    Average 96 stars, based on 286 article reviews
    Price from $9.99 to $1999.99
    cd69 monoclonal antibody - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    99
    Millipore expression gapdh
    ARL3-Specific Release Localizes LCK to the Immune Synapse (A and F) T:B cell conjugates stained with anti ARL3, LCK, or ARL13B antibodies as indicated in the presence or absence of SEE. (B) LR of ARL3, ARL13B, and LCK of conjugates from (A) and (F): ARL3 shows no localization to the synapse in the presence (LR = 0.3) or absence of SEE (n = 38). LCK shows LR = 1.9 in the presence of SEE and 0.7 in its absence (n = 42; p ≤ 0.0001). ARL13B shows LR = 2 in the presence of SEE (n = 32) and 0.6 in its absence (n = 16; p ≤ 0.0001). (C) ARL3 WT GFP and the constitutively active ARL3 Q71L GFP were nucleofected into cells and T:B cell conjugates, in the presence of SEE, were stained with anti LCK antibodies. (D) LR of LCK of conjugates from (C). ARL3 WT GFP expression had little effect on LR of LCK (n = 18) compared to cells not expressing ARL3 WT GFP (LR = 2). ARL3 Q71L GFP-expressing cells showed LR of LCK = 1 (n = 16, p = 0.0002). Differences in LR were analyzed using the Mann-Whitney U test (B and D). (E) <t>CD69</t> expression was analyzed via flow cytometry for cells stimulated with anti CD28 and CD3 antibodies and overexpressing ARL2-GFP, ARL2 Q70L GFP, ARL3 Q71L GFP, ARL3-GFP, or GFP. ARL3 Q71L GFP (p ≤ 0.0001), ARL3 WT GFP (p = 0.004), ARL2 WT GFP (p = 0.02), and ARL2 Q70L GFP (p = 0.03) p values refer to unpaired t test data. (G) Ciliated RPE cell stained with anti ARL13B antibody. Scale bars, 5 μm.
    Expression Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression gapdh/product/Millipore
    Average 99 stars, based on 124 article reviews
    Price from $9.99 to $1999.99
    expression gapdh - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    R&D Systems ccr7 expression
    Kinetics and activation status of immune-cell subsets throughout the infection. a – c Frequency (left set of plots) of CD4 and CD8 T cell subsets (CD45RA + <t>CCR7</t> + : naïve (N), CD45RA + CCR7 − : central memory (CM), CD45RA − CCR7 − : effector memory (EM), CD45RA + CCR7 − : terminal effector (TE)) ( a ), activated CD38 + HLADR + ( b ), and exhausted PD1 + CD57 + CD4 and CD8 T cells ( c ). d Frequency of γδ T cells (gated on CD3 + T cells) and CD16 and NKG2A expression (gated on γδ CD3 T cells). e Frequency of B cell subsets (CD21 + CD27 − : naïve, CD21 + CD27 + : resting memory (RM), CD21 − CD27 + : activated memory (AM), CD21 − CD27 − : exhausted (Ex)) and plasmablasts (CD38 ++ CD27 + ) gated on CD19 + B cells. f – h Frequency of NK-cell subsets (gated on CD3 − ) (CD56 Bright: CD56 ++ CD16 + , CD56dim: CD56 + CD16 + ) ( f ), differentiated Ki67 + NK cells (gated on CD56 dim CD57 + NK cells) ( g ) and inhibitor receptor NKG2A (gated on NK cells) ( h ). i Monocyte subsets (gated CD3 − CD56 − ) (classical monocytes: CD14 + CD16 − , intermediate monocytes: CD16 + CD14 + , non-classical monocytes: CD14 − CD16 + ) detected by flow cytometry of blood collected at days 14–20 following symptom onset from the patient and healthy donors ( n = 5, median with interquartile range); gating examples shown to the right
    Ccr7 Expression, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ccr7 expression/product/R&D Systems
    Average 95 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    ccr7 expression - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Millipore gapdh expression levels
    Kinetics and activation status of immune-cell subsets throughout the infection. a – c Frequency (left set of plots) of CD4 and CD8 T cell subsets (CD45RA + <t>CCR7</t> + : naïve (N), CD45RA + CCR7 − : central memory (CM), CD45RA − CCR7 − : effector memory (EM), CD45RA + CCR7 − : terminal effector (TE)) ( a ), activated CD38 + HLADR + ( b ), and exhausted PD1 + CD57 + CD4 and CD8 T cells ( c ). d Frequency of γδ T cells (gated on CD3 + T cells) and CD16 and NKG2A expression (gated on γδ CD3 T cells). e Frequency of B cell subsets (CD21 + CD27 − : naïve, CD21 + CD27 + : resting memory (RM), CD21 − CD27 + : activated memory (AM), CD21 − CD27 − : exhausted (Ex)) and plasmablasts (CD38 ++ CD27 + ) gated on CD19 + B cells. f – h Frequency of NK-cell subsets (gated on CD3 − ) (CD56 Bright: CD56 ++ CD16 + , CD56dim: CD56 + CD16 + ) ( f ), differentiated Ki67 + NK cells (gated on CD56 dim CD57 + NK cells) ( g ) and inhibitor receptor NKG2A (gated on NK cells) ( h ). i Monocyte subsets (gated CD3 − CD56 − ) (classical monocytes: CD14 + CD16 − , intermediate monocytes: CD16 + CD14 + , non-classical monocytes: CD14 − CD16 + ) detected by flow cytometry of blood collected at days 14–20 following symptom onset from the patient and healthy donors ( n = 5, median with interquartile range); gating examples shown to the right
    Gapdh Expression Levels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh expression levels/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gapdh expression levels - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Abcam gapdh expression
    Kinetics and activation status of immune-cell subsets throughout the infection. a – c Frequency (left set of plots) of CD4 and CD8 T cell subsets (CD45RA + <t>CCR7</t> + : naïve (N), CD45RA + CCR7 − : central memory (CM), CD45RA − CCR7 − : effector memory (EM), CD45RA + CCR7 − : terminal effector (TE)) ( a ), activated CD38 + HLADR + ( b ), and exhausted PD1 + CD57 + CD4 and CD8 T cells ( c ). d Frequency of γδ T cells (gated on CD3 + T cells) and CD16 and NKG2A expression (gated on γδ CD3 T cells). e Frequency of B cell subsets (CD21 + CD27 − : naïve, CD21 + CD27 + : resting memory (RM), CD21 − CD27 + : activated memory (AM), CD21 − CD27 − : exhausted (Ex)) and plasmablasts (CD38 ++ CD27 + ) gated on CD19 + B cells. f – h Frequency of NK-cell subsets (gated on CD3 − ) (CD56 Bright: CD56 ++ CD16 + , CD56dim: CD56 + CD16 + ) ( f ), differentiated Ki67 + NK cells (gated on CD56 dim CD57 + NK cells) ( g ) and inhibitor receptor NKG2A (gated on NK cells) ( h ). i Monocyte subsets (gated CD3 − CD56 − ) (classical monocytes: CD14 + CD16 − , intermediate monocytes: CD16 + CD14 + , non-classical monocytes: CD14 − CD16 + ) detected by flow cytometry of blood collected at days 14–20 following symptom onset from the patient and healthy donors ( n = 5, median with interquartile range); gating examples shown to the right
    Gapdh Expression, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh expression/product/Abcam
    Average 99 stars, based on 135 article reviews
    Price from $9.99 to $1999.99
    gapdh expression - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher cd274 expression
    Cytokine assay analysis of <t>CD274-mediated</t> effects of decidual T cell cytokine production. Graphs represent mean values of decidual T cell cytokine secretion from a minimum of five different patients. Error bars indicate SEM. PHA, phytohemagglutinin.
    Cd274 Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd274 expression/product/Thermo Fisher
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    cd274 expression - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    98
    Millipore ki 67 expression
    Histological characterization of large donor bile ducts from six patients that received a sex-mismatched liver graft. (A) Hematoxylin and eosin (H E) staining of the large donor bile ducts; the panels from left to right correspond to numbers 2, 4, and 6 in the tables. Patient #2 required a liver re-transplantation because of recurrence of hepatitis C. Relatively intact peribiliary glands (PBG) are shown in the left panel (arrowhead), some with debris in their lumen and detached epithelial cells. Patient #4 presented with a post-transplant cholangiopathy; disrupted luminal epithelium (arrow), infiltrative inflammatory cells, and a bile cast (arrowhead) were observed (central panel). Patient #6 was re-transplanted because of recurrence of primary sclerosing cholangitis (PSC). Arrowheads point toward damaged PBG (right panel). (B) Semiquantitative (SQ) score of destroyed PBG and inflammation around PBG. Patients #4, #5, and #6 presented with the highest number of destroyed PBG and inflammation around PBG. (C) Immunohistochemistry for cluster of differentiation 45 (CD45). The red dotted line encircles clusters of PBG. Inflammation was examined within this area. The inset is depicted from the larger image and shows PBG encircled by CD45+ inflammatory cells (arrowheads). Scale bars: 250 μm. (D) Immunohistochemistry for cytokeratin 19 (CK19), confirming the biliary origin of the PBG cells. Arrowheads point toward CK19 positive cells. (E) Immunohistochemistry for von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), and <t>Ki-67.</t> For all three stainings, a specified classifier was developed to determine the expression of vWF, α-SMA, and Ki-67 indicating the number of micro-vessels around PBG (i.e., microvascular density), myofibroblast activation, and proliferating cells (i.e., proliferation index), respectively (F) Relative expression of vWF, α-SMA, and Ki-67. Microvascular density appeared the highest in patient #2 and #6. Myofibroblast activation was most pronounced in patient #5. Patient #6 showed the highest proliferation index of the epithelial cell compartment.
    Ki 67 Expression, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ki 67 expression/product/Millipore
    Average 98 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    ki 67 expression - by Bioz Stars, 2020-09
    98/100 stars
      Buy from Supplier

    96
    Thermo Fisher opticho antibody express kit
    Histological characterization of large donor bile ducts from six patients that received a sex-mismatched liver graft. (A) Hematoxylin and eosin (H E) staining of the large donor bile ducts; the panels from left to right correspond to numbers 2, 4, and 6 in the tables. Patient #2 required a liver re-transplantation because of recurrence of hepatitis C. Relatively intact peribiliary glands (PBG) are shown in the left panel (arrowhead), some with debris in their lumen and detached epithelial cells. Patient #4 presented with a post-transplant cholangiopathy; disrupted luminal epithelium (arrow), infiltrative inflammatory cells, and a bile cast (arrowhead) were observed (central panel). Patient #6 was re-transplanted because of recurrence of primary sclerosing cholangitis (PSC). Arrowheads point toward damaged PBG (right panel). (B) Semiquantitative (SQ) score of destroyed PBG and inflammation around PBG. Patients #4, #5, and #6 presented with the highest number of destroyed PBG and inflammation around PBG. (C) Immunohistochemistry for cluster of differentiation 45 (CD45). The red dotted line encircles clusters of PBG. Inflammation was examined within this area. The inset is depicted from the larger image and shows PBG encircled by CD45+ inflammatory cells (arrowheads). Scale bars: 250 μm. (D) Immunohistochemistry for cytokeratin 19 (CK19), confirming the biliary origin of the PBG cells. Arrowheads point toward CK19 positive cells. (E) Immunohistochemistry for von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), and <t>Ki-67.</t> For all three stainings, a specified classifier was developed to determine the expression of vWF, α-SMA, and Ki-67 indicating the number of micro-vessels around PBG (i.e., microvascular density), myofibroblast activation, and proliferating cells (i.e., proliferation index), respectively (F) Relative expression of vWF, α-SMA, and Ki-67. Microvascular density appeared the highest in patient #2 and #6. Myofibroblast activation was most pronounced in patient #5. Patient #6 showed the highest proliferation index of the epithelial cell compartment.
    Opticho Antibody Express Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/opticho antibody express kit/product/Thermo Fisher
    Average 96 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    opticho antibody express kit - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    97
    Abcam protein expression
    Histological characterization of large donor bile ducts from six patients that received a sex-mismatched liver graft. (A) Hematoxylin and eosin (H E) staining of the large donor bile ducts; the panels from left to right correspond to numbers 2, 4, and 6 in the tables. Patient #2 required a liver re-transplantation because of recurrence of hepatitis C. Relatively intact peribiliary glands (PBG) are shown in the left panel (arrowhead), some with debris in their lumen and detached epithelial cells. Patient #4 presented with a post-transplant cholangiopathy; disrupted luminal epithelium (arrow), infiltrative inflammatory cells, and a bile cast (arrowhead) were observed (central panel). Patient #6 was re-transplanted because of recurrence of primary sclerosing cholangitis (PSC). Arrowheads point toward damaged PBG (right panel). (B) Semiquantitative (SQ) score of destroyed PBG and inflammation around PBG. Patients #4, #5, and #6 presented with the highest number of destroyed PBG and inflammation around PBG. (C) Immunohistochemistry for cluster of differentiation 45 (CD45). The red dotted line encircles clusters of PBG. Inflammation was examined within this area. The inset is depicted from the larger image and shows PBG encircled by CD45+ inflammatory cells (arrowheads). Scale bars: 250 μm. (D) Immunohistochemistry for cytokeratin 19 (CK19), confirming the biliary origin of the PBG cells. Arrowheads point toward CK19 positive cells. (E) Immunohistochemistry for von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), and <t>Ki-67.</t> For all three stainings, a specified classifier was developed to determine the expression of vWF, α-SMA, and Ki-67 indicating the number of micro-vessels around PBG (i.e., microvascular density), myofibroblast activation, and proliferating cells (i.e., proliferation index), respectively (F) Relative expression of vWF, α-SMA, and Ki-67. Microvascular density appeared the highest in patient #2 and #6. Myofibroblast activation was most pronounced in patient #5. Patient #6 showed the highest proliferation index of the epithelial cell compartment.
    Protein Expression, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein expression/product/Abcam
    Average 97 stars, based on 212 article reviews
    Price from $9.99 to $1999.99
    protein expression - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    93
    Thermo Fisher anti cd30 antibody expressing cell lines
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Anti Cd30 Antibody Expressing Cell Lines, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cd30 antibody expressing cell lines/product/Thermo Fisher
    Average 93 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    anti cd30 antibody expressing cell lines - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    89
    Becton Dickinson antibody expression
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Antibody Expression, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody expression/product/Becton Dickinson
    Average 89 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    antibody expression - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    89
    Thermo Fisher antibody expression
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Antibody Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody expression/product/Thermo Fisher
    Average 89 stars, based on 174 article reviews
    Price from $9.99 to $1999.99
    antibody expression - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    99
    Millipore anti beta actin antibody mouse monoclonal
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Anti Beta Actin Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti beta actin antibody mouse monoclonal/product/Millipore
    Average 99 stars, based on 8502 article reviews
    Price from $9.99 to $1999.99
    anti beta actin antibody mouse monoclonal - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    85
    4Gene anti inflammatory cytokine il 4 gene expression
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Anti Inflammatory Cytokine Il 4 Gene Expression, supplied by 4Gene, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti inflammatory cytokine il 4 gene expression/product/4Gene
    Average 85 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    anti inflammatory cytokine il 4 gene expression - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    92
    Abcam anti neural precursor cell
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Anti Neural Precursor Cell, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti neural precursor cell/product/Abcam
    Average 92 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    anti neural precursor cell - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti green fluorescent protein gfp antibody
    Molecular mechanism of <t>anti‐CD30‐LDM‐mediated</t> cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P
    Monoclonal Anti Green Fluorescent Protein Gfp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 439 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti green fluorescent protein gfp antibody/product/Millipore
    Average 99 stars, based on 439 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti green fluorescent protein gfp antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Abcam gfp expression
    Doxycycline-mediated gene regulation in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats . (A-F) Doxycycline-controlled EGFP expression in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-luc rats was visualized by immunohistochemistry on sagittal sections using an antibody against EGFP. (A-C) In the absence of Dox, strong but mosaic EGFP expression is found in the cortex and hippocampus (HC). (D-F) In adult rats, EGFP expression could be completely inhibited by chronic Dox treatment (1 mg/mL drinking water) from conception until analyses. (G-I) Dual-label fluorescent immunohistochemistry of brain slices with the neuronal marker <t>NeuN</t> and EGFP. (I) Co-localization of EGFP and NeuN was frequently found in the CA1 region of the HC indicating Ptet-controlled reporter gene expression in the absence of Dox. (J) CaMKIIα-promoter controlled tTA activity was directly visualized by EGFP fluorescence on sagittal brain sections of double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats in the HC. Strong expression is mainly found in neurons of the CA1 and CA3 region. (K,L) Level of luciferase activity in different brain regions in the absence (-Dox, black) and presence of Dox (+Dox, 1 mg/mL, lightly shaded). The measured double transgenic animals were obtained by crossing the CaMKIIα-tTA line 4.5 to the EGFP-Ptetbi-luc 66.1 reporter line. (K) Animals were treated with Dox from conception throughout life until the day of analyses at the age of two months (+Dox, n = 5). Untreated control animals were measured at the same age (-Dox, n = 8). In Dox-treated CaMKIIα-tTA/EGFP-Ptetbi-luc rats, virtually no luciferase activity could be detected, while strong luciferase activity was found in all forebrain-specific regions (cortex, HC, olfactory bulb) of untreated rats (-Dox), leading to a highly significant difference between Dox-treated and untreated rats (*** P ≤ 0.001). (L) To assess whether reporter gene expression could be suppressed with Dox in adult rats (+Dox adult), double transgenic animals, which had previously not received Dox, were treated with Dox for a period of three weeks during adulthood (-Dox, n = 10, +Dox adult, n = 6). Administration of Dox during adulthood suppressed luciferase activity, leading to a significant difference compared to untreated rats (*** P ≤ 0.001). All data are presented as mean values + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Stars represent P -values obtained by unpaired t-test. *** P ≤ 0.001. Light units are normalized to the protein content of the lysates. Scale bar: 100 μm. Dox: doxycycline hydrochloride; EGFP: enhanced green fluorescent protein; HC: hippocampus.
    Gfp Expression, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp expression/product/Abcam
    Average 99 stars, based on 302 article reviews
    Price from $9.99 to $1999.99
    gfp expression - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc β tubulin expression
    Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. <t>β-Tubulin</t> was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays
    β Tubulin Expression, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β tubulin expression/product/Cell Signaling Technology Inc
    Average 99 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    β tubulin expression - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc expression antibodies
    Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. <t>β-Tubulin</t> was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays
    Expression Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression antibodies/product/Cell Signaling Technology Inc
    Average 94 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    expression antibodies - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Sec63 inhibits IRE1α clustering during ER stress in cells. ( A ) HEK293 IRE1α−/− cells complemented with IRE1α-HA were transfected with either control, Sec63, or Sec62 siRNA. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 30h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and processed for immunostaining with anti-HA antibodies for IRE1α. Scale bars are 10 μm. ( B ) Quantification of the number of cells with IRE1α clusters from the panel A. Error bar represents standard deviation (n=2). ( C ) HEK293 IRE1α−/− cells were treated with the indicated siRNAs. After 30h of transfection, the cells were harvested and analyzed by immunoblotting for the indicated antigens. ( D ) HEK293 IRE1α−/− cells expressing either IRE1α-HA or IRE1α-CNX-TMD-HA were induced with 5ng/ml doxycycline and treated with either Tg or Tm for the indicated time points. The cells were then processed for immunostaining as in A. ( E ) The number of cells with IRE1α clusters were quantified from the panel D. Error bar represents standard deviation (n=2). ( F ) HEK293 IRE1α−/− cells expressing IRE1α-HA were transfected with either empty vector, wild type (WT) Sec63 or the J-domain mutant of Sec63. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 24h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and subsequently processed for immunostaining with anti-HA antibodies for IRE1α. ( G ) Quantification of the number of cells with IRE1α clusters from the panel F. Error bar represents standard deviation (n=2) ( H ) The cells were transfected as in the panel F, but analyzed by immunoblotting for the indicated antigens.

    Journal: bioRxiv

    Article Title: The Sec63/BiP complex suppresses higher-order oligomerization and RNase activity of IRE1α during ER stress

    doi: 10.1101/2020.04.03.024356

    Figure Lengend Snippet: Sec63 inhibits IRE1α clustering during ER stress in cells. ( A ) HEK293 IRE1α−/− cells complemented with IRE1α-HA were transfected with either control, Sec63, or Sec62 siRNA. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 30h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and processed for immunostaining with anti-HA antibodies for IRE1α. Scale bars are 10 μm. ( B ) Quantification of the number of cells with IRE1α clusters from the panel A. Error bar represents standard deviation (n=2). ( C ) HEK293 IRE1α−/− cells were treated with the indicated siRNAs. After 30h of transfection, the cells were harvested and analyzed by immunoblotting for the indicated antigens. ( D ) HEK293 IRE1α−/− cells expressing either IRE1α-HA or IRE1α-CNX-TMD-HA were induced with 5ng/ml doxycycline and treated with either Tg or Tm for the indicated time points. The cells were then processed for immunostaining as in A. ( E ) The number of cells with IRE1α clusters were quantified from the panel D. Error bar represents standard deviation (n=2). ( F ) HEK293 IRE1α−/− cells expressing IRE1α-HA were transfected with either empty vector, wild type (WT) Sec63 or the J-domain mutant of Sec63. The expression of IRE1α-HA was induced with 5ng/ml doxycycline. After 24h of transfection, the cells were either left untreated or treated with 5μg/ml Tg for 1.5h and subsequently processed for immunostaining with anti-HA antibodies for IRE1α. ( G ) Quantification of the number of cells with IRE1α clusters from the panel F. Error bar represents standard deviation (n=2) ( H ) The cells were transfected as in the panel F, but analyzed by immunoblotting for the indicated antigens.

    Article Snippet: The colonies were picked and the protein expression was evaluated by immunoblotting.

    Techniques: Transfection, Expressing, Immunostaining, Standard Deviation, Plasmid Preparation, Mutagenesis

    Short-term treatment with rIL-2/DTx leads to transient T reg depletion. ( A ) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry at the time indicated. ( B ) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry 7 d after the final dose. ( C and D ) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10 3 metacyclic L. major promastigotes. Lesional T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry ( C ), and IgG 1 antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples ( D ), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 ( A ), n = 4–6 ( B ) and n = 5–6 ( C and D ). ( A ) ANOVA P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Therapeutic Enhancement of Protective Immunity during Experimental Leishmaniasis

    doi: 10.1371/journal.pntd.0001316

    Figure Lengend Snippet: Short-term treatment with rIL-2/DTx leads to transient T reg depletion. ( A ) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry at the time indicated. ( B ) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry 7 d after the final dose. ( C and D ) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10 3 metacyclic L. major promastigotes. Lesional T reg (TCRβ + CD4 + CD25 + Foxp3 + cells) were quantified by flow cytometry ( C ), and IgG 1 antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples ( D ), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 ( A ), n = 4–6 ( B ) and n = 5–6 ( C and D ). ( A ) ANOVA P

    Article Snippet: After a further wash, cells were incubated with directly-conjugated monoclonal antibodies to TCR-β-FITC (H57-597), CD4-PE-Cy7 (RM4-5) or CD4-APC-Alexa Fluorochrome 750 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-PE (PC61), NK1.1-PerCp-Cy5.5 (PK136), CD49b-PE-Cy7 (DX5), F4/80-APC (BM8), CD11b-PerCp-Cy5.5 (M1/70), Gr-1-FITC (RB6-8C5), CD11c-Alexa Fluorochrome 700 (N418), B220-APC-Alexa Fluorochrome 750 (RA3-6B2), and/or CD19-PE (1D3) [all antibodies were from BD Biosciences and/or e-Bioscience] for 30 min. Quantification of Foxp3 expression was done using the Foxp3-APC (FJK-16s) staining kit (e-Bioscience) according to manufacturer's instructions, combined with directly-conjugated monoclonal antibodies TCR-β-FITC, CD4-APC-Alexa Fluorochrome 750 and CD25-PE (BD Biosciences and/or e-Bioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Infection, Enzyme-linked Immunosorbent Assay

    Combined therapy enhances resolution of experimental L. major infection in BALB/c mice. BALB/c mice were infected as in Figure 2 . ( A ) Lesion size. Beginning 30 d after infection, mice were treated: daily for 10 d with SSG (250 mg/kg; blue circles); 3 times at 5 d intervals with rIL-2/DTx (50 µg/kg; red circles); daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 µg/kg; black circles); or with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; open circles). ( B ) Lesional parasite burden; ( C ) Parasite burden in draining lymph nodes; ( D ) Parasite burden in liver (dotted line represents the limit of detection in the assay); ( E ) Parasite burden in spleen; ( F ) Splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + ) percentage; ( G ) Draining lymph node T reg percentage; and antigen-specific ( H ) IFN-γ, ( I ) IL-10 and ( J ) IL-4 secretion (by leukocytes isolated from lesional lymph nodes and cultured in presence of soluble Leishmania antigen) were quantified 45 d after infection. Data represent means +/− SE of 6–7 mice/group in a single experiment (with individual data points shown for parasite burden); ( A ) MANOVA P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Therapeutic Enhancement of Protective Immunity during Experimental Leishmaniasis

    doi: 10.1371/journal.pntd.0001316

    Figure Lengend Snippet: Combined therapy enhances resolution of experimental L. major infection in BALB/c mice. BALB/c mice were infected as in Figure 2 . ( A ) Lesion size. Beginning 30 d after infection, mice were treated: daily for 10 d with SSG (250 mg/kg; blue circles); 3 times at 5 d intervals with rIL-2/DTx (50 µg/kg; red circles); daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 µg/kg; black circles); or with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; open circles). ( B ) Lesional parasite burden; ( C ) Parasite burden in draining lymph nodes; ( D ) Parasite burden in liver (dotted line represents the limit of detection in the assay); ( E ) Parasite burden in spleen; ( F ) Splenic T reg (TCRβ + CD4 + CD25 + Foxp3 + ) percentage; ( G ) Draining lymph node T reg percentage; and antigen-specific ( H ) IFN-γ, ( I ) IL-10 and ( J ) IL-4 secretion (by leukocytes isolated from lesional lymph nodes and cultured in presence of soluble Leishmania antigen) were quantified 45 d after infection. Data represent means +/− SE of 6–7 mice/group in a single experiment (with individual data points shown for parasite burden); ( A ) MANOVA P

    Article Snippet: After a further wash, cells were incubated with directly-conjugated monoclonal antibodies to TCR-β-FITC (H57-597), CD4-PE-Cy7 (RM4-5) or CD4-APC-Alexa Fluorochrome 750 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-PE (PC61), NK1.1-PerCp-Cy5.5 (PK136), CD49b-PE-Cy7 (DX5), F4/80-APC (BM8), CD11b-PerCp-Cy5.5 (M1/70), Gr-1-FITC (RB6-8C5), CD11c-Alexa Fluorochrome 700 (N418), B220-APC-Alexa Fluorochrome 750 (RA3-6B2), and/or CD19-PE (1D3) [all antibodies were from BD Biosciences and/or e-Bioscience] for 30 min. Quantification of Foxp3 expression was done using the Foxp3-APC (FJK-16s) staining kit (e-Bioscience) according to manufacturer's instructions, combined with directly-conjugated monoclonal antibodies TCR-β-FITC, CD4-APC-Alexa Fluorochrome 750 and CD25-PE (BD Biosciences and/or e-Bioscience).

    Techniques: Infection, Mouse Assay, Isolation, Cell Culture

    ARL3-Specific Release Localizes LCK to the Immune Synapse (A and F) T:B cell conjugates stained with anti ARL3, LCK, or ARL13B antibodies as indicated in the presence or absence of SEE. (B) LR of ARL3, ARL13B, and LCK of conjugates from (A) and (F): ARL3 shows no localization to the synapse in the presence (LR = 0.3) or absence of SEE (n = 38). LCK shows LR = 1.9 in the presence of SEE and 0.7 in its absence (n = 42; p ≤ 0.0001). ARL13B shows LR = 2 in the presence of SEE (n = 32) and 0.6 in its absence (n = 16; p ≤ 0.0001). (C) ARL3 WT GFP and the constitutively active ARL3 Q71L GFP were nucleofected into cells and T:B cell conjugates, in the presence of SEE, were stained with anti LCK antibodies. (D) LR of LCK of conjugates from (C). ARL3 WT GFP expression had little effect on LR of LCK (n = 18) compared to cells not expressing ARL3 WT GFP (LR = 2). ARL3 Q71L GFP-expressing cells showed LR of LCK = 1 (n = 16, p = 0.0002). Differences in LR were analyzed using the Mann-Whitney U test (B and D). (E) CD69 expression was analyzed via flow cytometry for cells stimulated with anti CD28 and CD3 antibodies and overexpressing ARL2-GFP, ARL2 Q70L GFP, ARL3 Q71L GFP, ARL3-GFP, or GFP. ARL3 Q71L GFP (p ≤ 0.0001), ARL3 WT GFP (p = 0.004), ARL2 WT GFP (p = 0.02), and ARL2 Q70L GFP (p = 0.03) p values refer to unpaired t test data. (G) Ciliated RPE cell stained with anti ARL13B antibody. Scale bars, 5 μm.

    Journal: Developmental Cell

    Article Title: The Ciliary Machinery Is Repurposed for T Cell Immune Synapse Trafficking of LCK

    doi: 10.1016/j.devcel.2018.08.012

    Figure Lengend Snippet: ARL3-Specific Release Localizes LCK to the Immune Synapse (A and F) T:B cell conjugates stained with anti ARL3, LCK, or ARL13B antibodies as indicated in the presence or absence of SEE. (B) LR of ARL3, ARL13B, and LCK of conjugates from (A) and (F): ARL3 shows no localization to the synapse in the presence (LR = 0.3) or absence of SEE (n = 38). LCK shows LR = 1.9 in the presence of SEE and 0.7 in its absence (n = 42; p ≤ 0.0001). ARL13B shows LR = 2 in the presence of SEE (n = 32) and 0.6 in its absence (n = 16; p ≤ 0.0001). (C) ARL3 WT GFP and the constitutively active ARL3 Q71L GFP were nucleofected into cells and T:B cell conjugates, in the presence of SEE, were stained with anti LCK antibodies. (D) LR of LCK of conjugates from (C). ARL3 WT GFP expression had little effect on LR of LCK (n = 18) compared to cells not expressing ARL3 WT GFP (LR = 2). ARL3 Q71L GFP-expressing cells showed LR of LCK = 1 (n = 16, p = 0.0002). Differences in LR were analyzed using the Mann-Whitney U test (B and D). (E) CD69 expression was analyzed via flow cytometry for cells stimulated with anti CD28 and CD3 antibodies and overexpressing ARL2-GFP, ARL2 Q70L GFP, ARL3 Q71L GFP, ARL3-GFP, or GFP. ARL3 Q71L GFP (p ≤ 0.0001), ARL3 WT GFP (p = 0.004), ARL2 WT GFP (p = 0.02), and ARL2 Q70L GFP (p = 0.03) p values refer to unpaired t test data. (G) Ciliated RPE cell stained with anti ARL13B antibody. Scale bars, 5 μm.

    Article Snippet: Cells were stimulated for 4 hours and then analysed using flow cytometry to identify CD69 expression (CD69APC, Invitrogen, USA, MHCD6905).

    Techniques: Staining, Expressing, MANN-WHITNEY, Flow Cytometry, Cytometry

    Kinetics and activation status of immune-cell subsets throughout the infection. a – c Frequency (left set of plots) of CD4 and CD8 T cell subsets (CD45RA + CCR7 + : naïve (N), CD45RA + CCR7 − : central memory (CM), CD45RA − CCR7 − : effector memory (EM), CD45RA + CCR7 − : terminal effector (TE)) ( a ), activated CD38 + HLADR + ( b ), and exhausted PD1 + CD57 + CD4 and CD8 T cells ( c ). d Frequency of γδ T cells (gated on CD3 + T cells) and CD16 and NKG2A expression (gated on γδ CD3 T cells). e Frequency of B cell subsets (CD21 + CD27 − : naïve, CD21 + CD27 + : resting memory (RM), CD21 − CD27 + : activated memory (AM), CD21 − CD27 − : exhausted (Ex)) and plasmablasts (CD38 ++ CD27 + ) gated on CD19 + B cells. f – h Frequency of NK-cell subsets (gated on CD3 − ) (CD56 Bright: CD56 ++ CD16 + , CD56dim: CD56 + CD16 + ) ( f ), differentiated Ki67 + NK cells (gated on CD56 dim CD57 + NK cells) ( g ) and inhibitor receptor NKG2A (gated on NK cells) ( h ). i Monocyte subsets (gated CD3 − CD56 − ) (classical monocytes: CD14 + CD16 − , intermediate monocytes: CD16 + CD14 + , non-classical monocytes: CD14 − CD16 + ) detected by flow cytometry of blood collected at days 14–20 following symptom onset from the patient and healthy donors ( n = 5, median with interquartile range); gating examples shown to the right

    Journal: Journal of Clinical Immunology

    Article Title: Immune Alterations in a Patient with SARS-CoV-2-Related Acute Respiratory Distress Syndrome

    doi: 10.1007/s10875-020-00839-x

    Figure Lengend Snippet: Kinetics and activation status of immune-cell subsets throughout the infection. a – c Frequency (left set of plots) of CD4 and CD8 T cell subsets (CD45RA + CCR7 + : naïve (N), CD45RA + CCR7 − : central memory (CM), CD45RA − CCR7 − : effector memory (EM), CD45RA + CCR7 − : terminal effector (TE)) ( a ), activated CD38 + HLADR + ( b ), and exhausted PD1 + CD57 + CD4 and CD8 T cells ( c ). d Frequency of γδ T cells (gated on CD3 + T cells) and CD16 and NKG2A expression (gated on γδ CD3 T cells). e Frequency of B cell subsets (CD21 + CD27 − : naïve, CD21 + CD27 + : resting memory (RM), CD21 − CD27 + : activated memory (AM), CD21 − CD27 − : exhausted (Ex)) and plasmablasts (CD38 ++ CD27 + ) gated on CD19 + B cells. f – h Frequency of NK-cell subsets (gated on CD3 − ) (CD56 Bright: CD56 ++ CD16 + , CD56dim: CD56 + CD16 + ) ( f ), differentiated Ki67 + NK cells (gated on CD56 dim CD57 + NK cells) ( g ) and inhibitor receptor NKG2A (gated on NK cells) ( h ). i Monocyte subsets (gated CD3 − CD56 − ) (classical monocytes: CD14 + CD16 − , intermediate monocytes: CD16 + CD14 + , non-classical monocytes: CD14 − CD16 + ) detected by flow cytometry of blood collected at days 14–20 following symptom onset from the patient and healthy donors ( n = 5, median with interquartile range); gating examples shown to the right

    Article Snippet: CD4+ and CD8+ T cells were analyzed for CD45RA and CCR7 expression to identify the naive, memory, and effector cell subsets, for co-expression of activation (HLA-DR and CD38) and exhaustion/senescence (CD57and PD1) markers.

    Techniques: Activation Assay, Infection, Expressing, Flow Cytometry

    Cytokine assay analysis of CD274-mediated effects of decidual T cell cytokine production. Graphs represent mean values of decidual T cell cytokine secretion from a minimum of five different patients. Error bars indicate SEM. PHA, phytohemagglutinin.

    Journal:

    Article Title: Expression and Function of PDCD1 at the Human Maternal-Fetal Interface 1

    doi: 10.1095/biolreprod.107.066324

    Figure Lengend Snippet: Cytokine assay analysis of CD274-mediated effects of decidual T cell cytokine production. Graphs represent mean values of decidual T cell cytokine secretion from a minimum of five different patients. Error bars indicate SEM. PHA, phytohemagglutinin.

    Article Snippet: To ensure consistency of the presence (Jar/B7) and absence (Jar/V) of CD274 expression, both cell lines were routinely evaluated by flow cytometry using a PE-conjugated anti-CD274 antibody (clone MIH1) and PE-conjugated Mouse IgG1κ isotype control (eBioscience).

    Techniques: Cytokine Assay

    A ) Representative histograms from flow cytometric analysis of B7-H1 expression in control (Jar/V) or CD274-transfected (Jar/B7) cells and primary chorionic trohpoblast cells (Primary CTB). B ) Analysis of CD274 effects on decidual T cell apoptosis. Graphs

    Journal:

    Article Title: Expression and Function of PDCD1 at the Human Maternal-Fetal Interface 1

    doi: 10.1095/biolreprod.107.066324

    Figure Lengend Snippet: A ) Representative histograms from flow cytometric analysis of B7-H1 expression in control (Jar/V) or CD274-transfected (Jar/B7) cells and primary chorionic trohpoblast cells (Primary CTB). B ) Analysis of CD274 effects on decidual T cell apoptosis. Graphs

    Article Snippet: To ensure consistency of the presence (Jar/B7) and absence (Jar/V) of CD274 expression, both cell lines were routinely evaluated by flow cytometry using a PE-conjugated anti-CD274 antibody (clone MIH1) and PE-conjugated Mouse IgG1κ isotype control (eBioscience).

    Techniques: Flow Cytometry, Expressing, Transfection, CtB Assay

    Histological characterization of large donor bile ducts from six patients that received a sex-mismatched liver graft. (A) Hematoxylin and eosin (H E) staining of the large donor bile ducts; the panels from left to right correspond to numbers 2, 4, and 6 in the tables. Patient #2 required a liver re-transplantation because of recurrence of hepatitis C. Relatively intact peribiliary glands (PBG) are shown in the left panel (arrowhead), some with debris in their lumen and detached epithelial cells. Patient #4 presented with a post-transplant cholangiopathy; disrupted luminal epithelium (arrow), infiltrative inflammatory cells, and a bile cast (arrowhead) were observed (central panel). Patient #6 was re-transplanted because of recurrence of primary sclerosing cholangitis (PSC). Arrowheads point toward damaged PBG (right panel). (B) Semiquantitative (SQ) score of destroyed PBG and inflammation around PBG. Patients #4, #5, and #6 presented with the highest number of destroyed PBG and inflammation around PBG. (C) Immunohistochemistry for cluster of differentiation 45 (CD45). The red dotted line encircles clusters of PBG. Inflammation was examined within this area. The inset is depicted from the larger image and shows PBG encircled by CD45+ inflammatory cells (arrowheads). Scale bars: 250 μm. (D) Immunohistochemistry for cytokeratin 19 (CK19), confirming the biliary origin of the PBG cells. Arrowheads point toward CK19 positive cells. (E) Immunohistochemistry for von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), and Ki-67. For all three stainings, a specified classifier was developed to determine the expression of vWF, α-SMA, and Ki-67 indicating the number of micro-vessels around PBG (i.e., microvascular density), myofibroblast activation, and proliferating cells (i.e., proliferation index), respectively (F) Relative expression of vWF, α-SMA, and Ki-67. Microvascular density appeared the highest in patient #2 and #6. Myofibroblast activation was most pronounced in patient #5. Patient #6 showed the highest proliferation index of the epithelial cell compartment.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Evidence for Recipient-Derived Cells in Peribiliary Glands and Biliary Epithelium of the Large Donor Bile Ducts After Liver Transplantation

    doi: 10.3389/fcell.2020.00693

    Figure Lengend Snippet: Histological characterization of large donor bile ducts from six patients that received a sex-mismatched liver graft. (A) Hematoxylin and eosin (H E) staining of the large donor bile ducts; the panels from left to right correspond to numbers 2, 4, and 6 in the tables. Patient #2 required a liver re-transplantation because of recurrence of hepatitis C. Relatively intact peribiliary glands (PBG) are shown in the left panel (arrowhead), some with debris in their lumen and detached epithelial cells. Patient #4 presented with a post-transplant cholangiopathy; disrupted luminal epithelium (arrow), infiltrative inflammatory cells, and a bile cast (arrowhead) were observed (central panel). Patient #6 was re-transplanted because of recurrence of primary sclerosing cholangitis (PSC). Arrowheads point toward damaged PBG (right panel). (B) Semiquantitative (SQ) score of destroyed PBG and inflammation around PBG. Patients #4, #5, and #6 presented with the highest number of destroyed PBG and inflammation around PBG. (C) Immunohistochemistry for cluster of differentiation 45 (CD45). The red dotted line encircles clusters of PBG. Inflammation was examined within this area. The inset is depicted from the larger image and shows PBG encircled by CD45+ inflammatory cells (arrowheads). Scale bars: 250 μm. (D) Immunohistochemistry for cytokeratin 19 (CK19), confirming the biliary origin of the PBG cells. Arrowheads point toward CK19 positive cells. (E) Immunohistochemistry for von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), and Ki-67. For all three stainings, a specified classifier was developed to determine the expression of vWF, α-SMA, and Ki-67 indicating the number of micro-vessels around PBG (i.e., microvascular density), myofibroblast activation, and proliferating cells (i.e., proliferation index), respectively (F) Relative expression of vWF, α-SMA, and Ki-67. Microvascular density appeared the highest in patient #2 and #6. Myofibroblast activation was most pronounced in patient #5. Patient #6 showed the highest proliferation index of the epithelial cell compartment.

    Article Snippet: To quantify vWF, α-SMA, and Ki-67 expression, we used QuPath v0.2.0 to develop an appropriate classifier for each staining ( ).

    Techniques: Staining, Transplantation Assay, Immunohistochemistry, Expressing, Activation Assay

    Molecular mechanism of anti‐CD30‐LDM‐mediated cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: Molecular mechanism of anti‐CD30‐LDM‐mediated cell apoptosis. Western blot presented the levels of apoptosis‐associated proteins (A, B). (A) Cells were exposed to 0.03 n m anti‐CD30‐LDP/LDM/anti‐CD30‐LDM, respectively, and samples were collected at the appointed time to analyze the levels of p21 WAP1/CIP1 , p53, and p‐p53. β‐Actin was used as a loading control. (B) Cells were exposed to the appointed concentration of anti‐CD30‐LDM for 12 h, and samples were collected to analyzes the levels of cleaved‐caspase 3 and PARP. β‐Tubulin was used as a loading control. (C) Induction of caspase‐3/7 activity in Karpas299 or L540 cells treated with anti‐CD30‐LDM for 12 h. Fold induction in caspase‐3/7 activity was determined as described in Materials and methods . Means and SDs of triplicate experiments are shown (* P

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: Western Blot, Concentration Assay, Activity Assay

    Receptor‐mediated internalization of anti‐CD30‐LDP by CD30 + lymphoma cells. Cells were treated at 4 °C with anti‐CD30‐LDP labeled with Alexa Fluor 488‐conjugated anti‐human IgG. Anti‐CD30‐LDP bound to CD30‐positive cell line L540 and Karpas299, while no signal was detected on CD30‐negative cell line Raji (A). Anti‐CD30‐LDP was internalized and trafficked to the lysosomes in L540 (B) and Karpas299 (C) cells at 37 °C for 24 h. Cell surface or intracellular anti‐CD30‐LDP was visualized by immunofluorescence confocal microscopy. Anti‐CD30‐LDP is shown in green, the lysosomal marker Lamp‐1 is shown in red, and the Hoechst‐stained nuclei are in blue. Colocalization of signals for anti‐CD30‐LDP with Lamp‐1 is shown in yellow. The data shown were representative of three independent experiments. Scale bar = 10 μm.

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: Receptor‐mediated internalization of anti‐CD30‐LDP by CD30 + lymphoma cells. Cells were treated at 4 °C with anti‐CD30‐LDP labeled with Alexa Fluor 488‐conjugated anti‐human IgG. Anti‐CD30‐LDP bound to CD30‐positive cell line L540 and Karpas299, while no signal was detected on CD30‐negative cell line Raji (A). Anti‐CD30‐LDP was internalized and trafficked to the lysosomes in L540 (B) and Karpas299 (C) cells at 37 °C for 24 h. Cell surface or intracellular anti‐CD30‐LDP was visualized by immunofluorescence confocal microscopy. Anti‐CD30‐LDP is shown in green, the lysosomal marker Lamp‐1 is shown in red, and the Hoechst‐stained nuclei are in blue. Colocalization of signals for anti‐CD30‐LDP with Lamp‐1 is shown in yellow. The data shown were representative of three independent experiments. Scale bar = 10 μm.

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: Labeling, Immunofluorescence, Confocal Microscopy, Marker, Staining

    The characterization and in vitro activity of anti‐CD30‐LDM. (A) Reverse‐phase HPLC analysis of enediyne‐integrated anti‐CD30‐LDM using a Vydac C4 300A column at 340 nm. (B) Binding affinity of anti‐CD30‐LDM and anti‐CD30‐LDP to Karpas299 cells by FACS. (C) Cell viability assay. Karpas299, SU‐DHL‐1, L540, and L428 cell lines were treated with anti‐CD30‐LDM and LDM in a series of concentrations (0.001–1 n m ) for 48 h. Cell viability was tested by Cell Counting Kit‐8 (CCK‐8). Results are the mean values ± SD of three replicates. (D) Flow cytometry analysis of apoptosis of L540 or Karpas299 cells treated with increasing concentrations of anti‐CD30‐LDM for 24 h, respectively. (E) Cell cycle arrest assay of Karpas299 or L540 cells by flow cytometry. Cells were treated with indicated concentrations of anti‐CD30‐LDM for 24 h.

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: The characterization and in vitro activity of anti‐CD30‐LDM. (A) Reverse‐phase HPLC analysis of enediyne‐integrated anti‐CD30‐LDM using a Vydac C4 300A column at 340 nm. (B) Binding affinity of anti‐CD30‐LDM and anti‐CD30‐LDP to Karpas299 cells by FACS. (C) Cell viability assay. Karpas299, SU‐DHL‐1, L540, and L428 cell lines were treated with anti‐CD30‐LDM and LDM in a series of concentrations (0.001–1 n m ) for 48 h. Cell viability was tested by Cell Counting Kit‐8 (CCK‐8). Results are the mean values ± SD of three replicates. (D) Flow cytometry analysis of apoptosis of L540 or Karpas299 cells treated with increasing concentrations of anti‐CD30‐LDM for 24 h, respectively. (E) Cell cycle arrest assay of Karpas299 or L540 cells by flow cytometry. Cells were treated with indicated concentrations of anti‐CD30‐LDM for 24 h.

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: In Vitro, Activity Assay, High Performance Liquid Chromatography, Binding Assay, FACS, Viability Assay, Cell Counting, CCK-8 Assay, Flow Cytometry, Cytometry

    In vivo efficacy of anti‐CD30‐LDM against Karpas299 xenograft model. (A) The antitumor effects of anti‐CD30‐LDM in NOD/SCID mice bearing Karpas299 xenografts ( n = 6). The agents were administered at doses indicated in the figure on a Q7D × 2 schedule, and arrows indicate days of administration. * P

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: In vivo efficacy of anti‐CD30‐LDM against Karpas299 xenograft model. (A) The antitumor effects of anti‐CD30‐LDM in NOD/SCID mice bearing Karpas299 xenografts ( n = 6). The agents were administered at doses indicated in the figure on a Q7D × 2 schedule, and arrows indicate days of administration. * P

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: In Vivo, Mouse Assay

    Anti‐CD30‐LDP retains binding and functional properties of parent anti‐CD30 antibody in vitro . (A) Binding activity of anti‐CD30‐LDP and anti‐CD30 antibody to recombinant human CD30 by ELISA. (B) Surface plasmon resonance (SPR) sensorgrams of anti‐CD30‐LDP binding with CD30 antigen. Various concentrations of antigen (human recombinant CD30, 2.03‐65 n m ) were applied on CM5 chip with immobilized anti‐CD30‐LDP fusion protein. Each sensorgram represents different concentrations of CD30. The CD30 at 2.03 n m was injected twice to verify reproducibility. (C) Western blot analysis of CD30 expression levels on different cancer cells. (D) Binding activity to different cell lines. The cells were incubated with anti‐CD30‐LDP of 10 μg·mL −1 and FITC‐conjugated anti‐human Fc second antibody, and then, cell‐bound fluorescence was determined by FACS analysis. The horizontal axis represents the values of MFI. (E) Binding curves of increasing concentrations of anti‐CD30‐LDP (0.001‐3 μg·mL −1 ) to different cancer cells by FACS analysis. The vertical axis represents the MFI values. (F) In vitro ADCC analysis of anti‐CD30‐LDP and anti‐CD30 antibody. The ADCC assay was performed using PBMCs as effector cells and Karpas299 or L540 cells as target cells at a ratio of 40 : 1. The concentration of anti‐CD30‐LDP was 10 μg·mL −1 . Meanwhile, the cetuximab and the target cell line A431 were used as a positive control.

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: Anti‐CD30‐LDP retains binding and functional properties of parent anti‐CD30 antibody in vitro . (A) Binding activity of anti‐CD30‐LDP and anti‐CD30 antibody to recombinant human CD30 by ELISA. (B) Surface plasmon resonance (SPR) sensorgrams of anti‐CD30‐LDP binding with CD30 antigen. Various concentrations of antigen (human recombinant CD30, 2.03‐65 n m ) were applied on CM5 chip with immobilized anti‐CD30‐LDP fusion protein. Each sensorgram represents different concentrations of CD30. The CD30 at 2.03 n m was injected twice to verify reproducibility. (C) Western blot analysis of CD30 expression levels on different cancer cells. (D) Binding activity to different cell lines. The cells were incubated with anti‐CD30‐LDP of 10 μg·mL −1 and FITC‐conjugated anti‐human Fc second antibody, and then, cell‐bound fluorescence was determined by FACS analysis. The horizontal axis represents the values of MFI. (E) Binding curves of increasing concentrations of anti‐CD30‐LDP (0.001‐3 μg·mL −1 ) to different cancer cells by FACS analysis. The vertical axis represents the MFI values. (F) In vitro ADCC analysis of anti‐CD30‐LDP and anti‐CD30 antibody. The ADCC assay was performed using PBMCs as effector cells and Karpas299 or L540 cells as target cells at a ratio of 40 : 1. The concentration of anti‐CD30‐LDP was 10 μg·mL −1 . Meanwhile, the cetuximab and the target cell line A431 were used as a positive control.

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: Binding Assay, Functional Assay, In Vitro, Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, SPR Assay, Chromatin Immunoprecipitation, Injection, Western Blot, Expressing, Incubation, Fluorescence, FACS, ADCC Assay, Concentration Assay, Positive Control

    Targeting of fluorescently labeled proteins to established L540 and Karpas299 xenografts in NOD/SCID mice. Anti‐CD30‐LDP (A) or free LDP (B) was labeled with DyLight 680 and injected into tumor‐bearing NOD/SCID mice through tail veins at 20 mg·kg −1 , respectively. In vivo fluorescence images were taken at appointed time. Color scale represents photons·s −1 ·cm −2 ·steradian −1 .

    Journal: Molecular Oncology

    Article Title: A novel enediyne‐integrated antibody–drug conjugate shows promising antitumor efficacy against CD30+ lymphomas

    doi: 10.1002/1878-0261.12166

    Figure Lengend Snippet: Targeting of fluorescently labeled proteins to established L540 and Karpas299 xenografts in NOD/SCID mice. Anti‐CD30‐LDP (A) or free LDP (B) was labeled with DyLight 680 and injected into tumor‐bearing NOD/SCID mice through tail veins at 20 mg·kg −1 , respectively. In vivo fluorescence images were taken at appointed time. Color scale represents photons·s −1 ·cm −2 ·steradian −1 .

    Article Snippet: For the generation of anti‐CD30‐LDP and anti‐CD30 antibody‐expressing cell lines, pIZDHL‐anti‐CD30‐LDP and pIZDHL‐anti‐CD30 were linearized and transfected into CHO/dhFr‐ cells by lipofectin transfection (Invitrogen, Carlsbad, CA, USA), respectively.

    Techniques: Labeling, Mouse Assay, Injection, In Vivo, Fluorescence

    Doxycycline-mediated gene regulation in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats . (A-F) Doxycycline-controlled EGFP expression in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-luc rats was visualized by immunohistochemistry on sagittal sections using an antibody against EGFP. (A-C) In the absence of Dox, strong but mosaic EGFP expression is found in the cortex and hippocampus (HC). (D-F) In adult rats, EGFP expression could be completely inhibited by chronic Dox treatment (1 mg/mL drinking water) from conception until analyses. (G-I) Dual-label fluorescent immunohistochemistry of brain slices with the neuronal marker NeuN and EGFP. (I) Co-localization of EGFP and NeuN was frequently found in the CA1 region of the HC indicating Ptet-controlled reporter gene expression in the absence of Dox. (J) CaMKIIα-promoter controlled tTA activity was directly visualized by EGFP fluorescence on sagittal brain sections of double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats in the HC. Strong expression is mainly found in neurons of the CA1 and CA3 region. (K,L) Level of luciferase activity in different brain regions in the absence (-Dox, black) and presence of Dox (+Dox, 1 mg/mL, lightly shaded). The measured double transgenic animals were obtained by crossing the CaMKIIα-tTA line 4.5 to the EGFP-Ptetbi-luc 66.1 reporter line. (K) Animals were treated with Dox from conception throughout life until the day of analyses at the age of two months (+Dox, n = 5). Untreated control animals were measured at the same age (-Dox, n = 8). In Dox-treated CaMKIIα-tTA/EGFP-Ptetbi-luc rats, virtually no luciferase activity could be detected, while strong luciferase activity was found in all forebrain-specific regions (cortex, HC, olfactory bulb) of untreated rats (-Dox), leading to a highly significant difference between Dox-treated and untreated rats (*** P ≤ 0.001). (L) To assess whether reporter gene expression could be suppressed with Dox in adult rats (+Dox adult), double transgenic animals, which had previously not received Dox, were treated with Dox for a period of three weeks during adulthood (-Dox, n = 10, +Dox adult, n = 6). Administration of Dox during adulthood suppressed luciferase activity, leading to a significant difference compared to untreated rats (*** P ≤ 0.001). All data are presented as mean values + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Stars represent P -values obtained by unpaired t-test. *** P ≤ 0.001. Light units are normalized to the protein content of the lysates. Scale bar: 100 μm. Dox: doxycycline hydrochloride; EGFP: enhanced green fluorescent protein; HC: hippocampus.

    Journal: BMC Biology

    Article Title: Conditional gene expression systems in the transgenic rat brain

    doi: 10.1186/1741-7007-10-77

    Figure Lengend Snippet: Doxycycline-mediated gene regulation in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats . (A-F) Doxycycline-controlled EGFP expression in double transgenic CaMKIIα-tTA/EGFP-Ptetbi-luc rats was visualized by immunohistochemistry on sagittal sections using an antibody against EGFP. (A-C) In the absence of Dox, strong but mosaic EGFP expression is found in the cortex and hippocampus (HC). (D-F) In adult rats, EGFP expression could be completely inhibited by chronic Dox treatment (1 mg/mL drinking water) from conception until analyses. (G-I) Dual-label fluorescent immunohistochemistry of brain slices with the neuronal marker NeuN and EGFP. (I) Co-localization of EGFP and NeuN was frequently found in the CA1 region of the HC indicating Ptet-controlled reporter gene expression in the absence of Dox. (J) CaMKIIα-promoter controlled tTA activity was directly visualized by EGFP fluorescence on sagittal brain sections of double transgenic CaMKIIα-tTA/EGFP-Ptetbi-Luc rats in the HC. Strong expression is mainly found in neurons of the CA1 and CA3 region. (K,L) Level of luciferase activity in different brain regions in the absence (-Dox, black) and presence of Dox (+Dox, 1 mg/mL, lightly shaded). The measured double transgenic animals were obtained by crossing the CaMKIIα-tTA line 4.5 to the EGFP-Ptetbi-luc 66.1 reporter line. (K) Animals were treated with Dox from conception throughout life until the day of analyses at the age of two months (+Dox, n = 5). Untreated control animals were measured at the same age (-Dox, n = 8). In Dox-treated CaMKIIα-tTA/EGFP-Ptetbi-luc rats, virtually no luciferase activity could be detected, while strong luciferase activity was found in all forebrain-specific regions (cortex, HC, olfactory bulb) of untreated rats (-Dox), leading to a highly significant difference between Dox-treated and untreated rats (*** P ≤ 0.001). (L) To assess whether reporter gene expression could be suppressed with Dox in adult rats (+Dox adult), double transgenic animals, which had previously not received Dox, were treated with Dox for a period of three weeks during adulthood (-Dox, n = 10, +Dox adult, n = 6). Administration of Dox during adulthood suppressed luciferase activity, leading to a significant difference compared to untreated rats (*** P ≤ 0.001). All data are presented as mean values + standard error of the mean. Logarithmic data transformation was performed prior to statistical analysis. Stars represent P -values obtained by unpaired t-test. *** P ≤ 0.001. Light units are normalized to the protein content of the lysates. Scale bar: 100 μm. Dox: doxycycline hydrochloride; EGFP: enhanced green fluorescent protein; HC: hippocampus.

    Article Snippet: Double fluorescence IHC was performed to visualize β-gal and GFP expression in NeuN-positive neurons using the primary antibodies chicken anti-β-gal (Abcam, Cambridge, UK, 1:10,000), mouse monoclonal anti-NeuN (Millipore, Schwalbach, Germany 1:4,000) and polyclonal rabbit anti-GFP (Invitrogen, 1:1,000).

    Techniques: Transgenic Assay, Expressing, Immunohistochemistry, Marker, Activity Assay, Fluorescence, Luciferase, Transformation Assay

    Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. β-Tubulin was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays

    Journal: Cell Death & Disease

    Article Title: Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway

    doi: 10.1038/s41419-017-0119-z

    Figure Lengend Snippet: Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. β-Tubulin was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays

    Article Snippet: The primary antibodies used in this study targeted the following proteins: Notch1, Hes1, Nestin, Tuj-1, CD133, and GFAP (Abcam, USA; dilution 1:1000); and NICD, NF-κB(p65), cyclin D1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 antibodies (Cell Signaling Technology (CST), USA; dilution 1:1000). β-Tubulin expression (CST; dilution 1:2000) was used as a loading control to normalize the results.

    Techniques: Transfection, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Immunoprecipitation

    Effect of DAPT on NF-κB(p65) expression in glioma cells a , b DAPT-induced apoptosis of glioma cells in vitro. The percentages of apoptotic cells were significantly increased after DAPT treatment. c Immunofluorescence shows Hes1 and p65 expression in glioma cells after DAPT treatment. The scale bar corresponds to 20 µm. d After DAPT treatment, the Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 expression levels were detected by western blotting. β-Tubulin was used as a loading control. * P

    Journal: Cell Death & Disease

    Article Title: Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway

    doi: 10.1038/s41419-017-0119-z

    Figure Lengend Snippet: Effect of DAPT on NF-κB(p65) expression in glioma cells a , b DAPT-induced apoptosis of glioma cells in vitro. The percentages of apoptotic cells were significantly increased after DAPT treatment. c Immunofluorescence shows Hes1 and p65 expression in glioma cells after DAPT treatment. The scale bar corresponds to 20 µm. d After DAPT treatment, the Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 expression levels were detected by western blotting. β-Tubulin was used as a loading control. * P

    Article Snippet: The primary antibodies used in this study targeted the following proteins: Notch1, Hes1, Nestin, Tuj-1, CD133, and GFAP (Abcam, USA; dilution 1:1000); and NICD, NF-κB(p65), cyclin D1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 antibodies (Cell Signaling Technology (CST), USA; dilution 1:1000). β-Tubulin expression (CST; dilution 1:2000) was used as a loading control to normalize the results.

    Techniques: Expressing, In Vitro, Immunofluorescence, Western Blot