Human Lymphotoxin  beta R/TNFRSF3 Antibody

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

LT beta R is internalized and trafficked towards degradation upon ligand binding. A549 cells were stimulated with Ago for the indicated time periods and immunostained for LT beta R, EEA1 and LAMP1 (a) or trans- (TGN46) and cis-Golgi (GM130) (b). Insets show magnified views of boxed regions in the main images. Scale bars, 20 μm. Graphs represent the analysis of colocalization between LT beta R and EEA1 or LAMP1, and integral intensity of LT beta R (a) and colocalization between LT beta R and GM130 or TGN46 (b). Data represent the means ± SEM, n ≥ 5 (a), n = 3 (b); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. c Lysates of A549 cells stimulated with Ago for different time periods were analyzed by Western blotting with antibodies against LT beta R and vinculin (used as a loading control). Representative blots are shown. Values below blots represent the averaged LT beta R/vinculin ratio (n = 5) in cells stimulated with Ago for the indicated time periods. Values are normalized to unstimulated control (time 0) set as 1.d Lysates of A549, HEK293T, CCD1070Sk and HeLa cells pretreated or not for 20 h (A549, HEK293T, CCD1070Sk) or 16 h (HeLa) with lysosomal degradation inhibitor, chloroquine (CQ), stimulated or not with Ago for the next 4 h were analyzed by Western blotting with antibodies against LT beta R and GAPDH (used as a loading control). Representative blots are shown. Table presents the fold change of LT beta R abundance in stimulated vs unstimulated cells (means, n ≥ 3) Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Clathrin and dynamin deficiency reduces activation of the non-canonical NF-kappa B pathway. a-c A549, HEK293T and CCD1070Sk cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) and stimulated or not with Ago for 1 h. d, f A549 cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) and stimulated or not with Ago for 1 h. e, g A549 cells were treated with dynasore (DYN) or DMSO and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Representative blots are shown. Values presented below blots represent the averaged p52/p100/loading control (a-e) or NIK/loading control ratio (f-g) from at least three experiments (normalized to the selected control, set as 1) in cells stimulated with Ago Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking dynamin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) or treated with dynasore (DYN, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (b, c, f), n = 4 (a, e, g); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of dynamin knock-down. Representative blots are shown Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking clathrin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) or treated with chlorpromazine (CPZ, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (a, b, f, g), n = 4 (c, e); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of clathrin knock-down. Representative blots are shown. The blots of GAPDH shown in panels b and c are also shown in panel d Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking dynamin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) or treated with dynasore (DYN, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (b, c, f), n = 4 (a, e, g); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of dynamin knock-down. Representative blots are shown Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking clathrin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) or treated with chlorpromazine (CPZ, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (a, b, f, g), n = 4 (c, e); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of clathrin knock-down. Representative blots are shown. The blots of GAPDH shown in panels b and c are also shown in panel d Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Clathrin and dynamin deficiency reduces activation of the non-canonical NF-kappa B pathway. a-c A549, HEK293T and CCD1070Sk cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) and stimulated or not with Ago for 1 h. d, f A549 cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) and stimulated or not with Ago for 1 h. e, g A549 cells were treated with dynasore (DYN) or DMSO and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Representative blots are shown. Values presented below blots represent the averaged p52/p100/loading control (a-e) or NIK/loading control ratio (f-g) from at least three experiments (normalized to the selected control, set as 1) in cells stimulated with Ago Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking clathrin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) or treated with chlorpromazine (CPZ, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (a, b, f, g), n = 4 (c, e); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of clathrin knock-down. Representative blots are shown. The blots of GAPDH shown in panels b and c are also shown in panel d Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

LT beta R is internalized and trafficked towards degradation upon ligand binding. A549 cells were stimulated with Ago for the indicated time periods and immunostained for LT beta R, EEA1 and LAMP1 (a) or trans- (TGN46) and cis-Golgi (GM130) (b). Insets show magnified views of boxed regions in the main images. Scale bars, 20 μm. Graphs represent the analysis of colocalization between LT beta R and EEA1 or LAMP1, and integral intensity of LT beta R (a) and colocalization between LT beta R and GM130 or TGN46 (b). Data represent the means ± SEM, n ≥ 5 (a), n = 3 (b); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. c Lysates of A549 cells stimulated with Ago for different time periods were analyzed by Western blotting with antibodies against LT beta R and vinculin (used as a loading control). Representative blots are shown. Values below blots represent the averaged LT beta R/vinculin ratio (n = 5) in cells stimulated with Ago for the indicated time periods. Values are normalized to unstimulated control (time 0) set as 1.d Lysates of A549, HEK293T, CCD1070Sk and HeLa cells pretreated or not for 20 h (A549, HEK293T, CCD1070Sk) or 16 h (HeLa) with lysosomal degradation inhibitor, chloroquine (CQ), stimulated or not with Ago for the next 4 h were analyzed by Western blotting with antibodies against LT beta R and GAPDH (used as a loading control). Representative blots are shown. Table presents the fold change of LT beta R abundance in stimulated vs unstimulated cells (means, n ≥ 3) Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Clathrin and dynamin deficiency reduces activation of the non-canonical NF-kappa B pathway. a-c A549, HEK293T and CCD1070Sk cells were transfected with siRNAs targeting clathrin (CHC) (two oligonucleotides) along with control, non-targeting siRNAs (two oligonucleotides) and stimulated or not with Ago for 1 h. d, f A549 cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) and stimulated or not with Ago for 1 h. e, g A549 cells were treated with dynasore (DYN) or DMSO and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Representative blots are shown. Values presented below blots represent the averaged p52/p100/loading control (a-e) or NIK/loading control ratio (f-g) from at least three experiments (normalized to the selected control, set as 1) in cells stimulated with Ago Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking dynamin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) or treated with dynasore (DYN, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (b, c, f), n = 4 (a, e, g); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of dynamin knock-down. Representative blots are shown Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Lymphotoxin beta R/TNFRSF3 by Western Blot

Blocking dynamin-dependent endocytosis enhances activation of canonical NF-kappa B signaling by LT beta R. A549 (a), HEK293T (b, d) and CCD1070Sk (c, d) cells were transfected with siRNAs targeting dynamin-1/2 (three combinations of oligonucleotides targeting dynamin-1 and dynamin-2, see Methods) along with non-targeting control (Ctrl) siRNAs (two combinations of oligonucleotides, see Methods) or treated with dynasore (DYN, e-g) along with DMSO, and stimulated or not with Ago for 1 h. Lysates of cells were analyzed by Western blotting with antibodies against the indicated proteins. Graphs show densitometric analysis of abundance of I kappa B alpha, normalized to loading controls (GAPDH or vinculin). Values are presented as a fold change vs unstimulated non-targeting controls – averaged non-targeting controls (AvCtrl) or DMSO, set as 1. Data represent the means ± SEM, n = 3 (b, c, f), n = 4 (a, e, g); ns - P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by one sample t test. Tables present the fold change of I kappa B alpha abundance in stimulated vs unstimulated cells (means, n ≥ 3). d HEK293T and CCD1070Sk cells were analyzed with respect to the efficiency of dynamin knock-down. Representative blots are shown Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33148272), licensed under a CC-BY license. Not internally tested by R&D Systems.