dock 8 Search Results


gene exp dock8 hs00298892 m1  (Thermo Fisher)
86
Thermo Fisher gene exp dock8 hs00298892 m1
Gene Exp Dock8 Hs00298892 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp dock8 hs00298892 m1 - by Bioz Stars, 2026-05
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rabbit anti dock8 antibody  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology rabbit anti dock8 antibody
(A) Immunoblots showing the phosphoinositide-binding specificity of <t>DOCK8.</t> Lysates of BW5147α − β − cells expressing HA-tagged wild-type (WT) DOCK8 were used as an input for incubation with lipid vesicles containing each phosphoinositide at indicated concentrations (%). Lipid-associated fractions were analyzed by immunoblotting with anti-HA antibody. Positions of the size marker were shown on the right. (B) Schematic diagram of DOCK8 constructs used in the experiments. (C) Immunoblots showing no detectable binding to PI(4,5)P2 of a DOCK8 mutant in which the DHR-1 domain is deleted (ΔDHR-1). Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8-ΔDHR-1 (bottom) were used as an input for lipid-binding assays. (D) Immunoblots showing PI(4,5)P2 binding of recombinant GST-fusion DOCK8 DHR-1 domain (top) and GST-fusion PLCδ1 PH domain (bottom).
Rabbit Anti Dock8 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti dock8 antibody/product/Santa Cruz Biotechnology
Average 92 stars, based on 1 article reviews
rabbit anti dock8 antibody - by Bioz Stars, 2026-05
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dock8  (OriGene)
94
OriGene dock8
(A) Immunoblots showing the phosphoinositide-binding specificity of <t>DOCK8.</t> Lysates of BW5147α − β − cells expressing HA-tagged wild-type (WT) DOCK8 were used as an input for incubation with lipid vesicles containing each phosphoinositide at indicated concentrations (%). Lipid-associated fractions were analyzed by immunoblotting with anti-HA antibody. Positions of the size marker were shown on the right. (B) Schematic diagram of DOCK8 constructs used in the experiments. (C) Immunoblots showing no detectable binding to PI(4,5)P2 of a DOCK8 mutant in which the DHR-1 domain is deleted (ΔDHR-1). Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8-ΔDHR-1 (bottom) were used as an input for lipid-binding assays. (D) Immunoblots showing PI(4,5)P2 binding of recombinant GST-fusion DOCK8 DHR-1 domain (top) and GST-fusion PLCδ1 PH domain (bottom).
Dock8, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dock8/product/OriGene
Average 94 stars, based on 1 article reviews
dock8 - by Bioz Stars, 2026-05
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dock 8  (Proteintech)
94
Proteintech dock 8
(A) Immunoblots showing the phosphoinositide-binding specificity of <t>DOCK8.</t> Lysates of BW5147α − β − cells expressing HA-tagged wild-type (WT) DOCK8 were used as an input for incubation with lipid vesicles containing each phosphoinositide at indicated concentrations (%). Lipid-associated fractions were analyzed by immunoblotting with anti-HA antibody. Positions of the size marker were shown on the right. (B) Schematic diagram of DOCK8 constructs used in the experiments. (C) Immunoblots showing no detectable binding to PI(4,5)P2 of a DOCK8 mutant in which the DHR-1 domain is deleted (ΔDHR-1). Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8-ΔDHR-1 (bottom) were used as an input for lipid-binding assays. (D) Immunoblots showing PI(4,5)P2 binding of recombinant GST-fusion DOCK8 DHR-1 domain (top) and GST-fusion PLCδ1 PH domain (bottom).
Dock 8, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dock 8/product/Proteintech
Average 94 stars, based on 1 article reviews
dock 8 - by Bioz Stars, 2026-05
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hmgb1 elisa kit  (Biorbyt)
93
Biorbyt hmgb1 elisa kit
Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release <t>HMGB1</t> as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.
Hmgb1 Elisa Kit, supplied by Biorbyt, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dock8-deficient gfp-expressing mice  (Jackson Laboratory)
90
Jackson Laboratory dock8-deficient gfp-expressing mice
Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release <t>HMGB1</t> as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.
Dock8 Deficient Gfp Expressing Mice, supplied by Jackson Laboratory, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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gst-conjugated dock8 amino acids 1–238  (Cocalico Inc)
90
Cocalico Inc gst-conjugated dock8 amino acids 1–238
Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release <t>HMGB1</t> as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.
Gst Conjugated Dock8 Amino Acids 1–238, supplied by Cocalico Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gst-conjugated dock8 amino acids 1–238/product/Cocalico Inc
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gst-conjugated dock8 amino acids 1–238 - by Bioz Stars, 2026-05
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dock8 antibodies  (ImmunoGen Inc)
90
ImmunoGen Inc dock8 antibodies
STAT3 phosphorylation analysis after stimulation. ( a ) Western blot analysis of whole cell lysates of PBMCs, unstimulated or 20 min. stimulated with 200 ng/ml IL6 or IL10. Expression of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 (STAT3) of the two affected siblings and a healthy control was assessed; Actin as loading control. ( b ) Representative flow cytometric analysis showing diminished Y705-STAT3 phosphorylation after 20 min. stimulation with 200 ng/ml IL6 (solid line) versus unremarkable results after stimulation with 20 ng/ml IL10 (dotted line) and 10 ng/ml IL21 (dashed line) in lymphocytes of patient II.2 compared to unremarkable results in patient II.3 and a healthy control; filled gray area: unstimulated lymphocytes. ( c ) Flow cytometric analysis showing Y705-STAT3 phosphorylation after 20 min. stimulation with 20 ng/ml IL6 (solid line) or IL10 (dotted line) and 10 ng/ml IL21 (dashed line) comparable to healthy control in lymphocytes of one (representative of four) <t>DOCK8-HIES</t> patient. ( d ) Restored STAT3 phosphorylation after IL6 stimulation (solid line) in patient II.2 15 months after HSCT compared to unstimulated (filled gray area) and IL10-stimulated (dotted line) lymphocytes.
Dock8 Antibodies, supplied by ImmunoGen Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dock8 antibodies/product/ImmunoGen Inc
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pyrosequencing of dock8  (Pyrosequencing Inc)
90
Pyrosequencing Inc pyrosequencing of dock8
A novel compound heterozygous mutation in <t>DOCK8</t> results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.
Pyrosequencing Of Dock8, supplied by Pyrosequencing Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pyrosequencing of dock8/product/Pyrosequencing Inc
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pyrosequencing of dock8 - by Bioz Stars, 2026-05
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small interfering rnas specific to dock8 sirna-dock8-2  (Shanghai Genechem Ltd)
90
Shanghai Genechem Ltd small interfering rnas specific to dock8 sirna-dock8-2
A novel compound heterozygous mutation in <t>DOCK8</t> results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.
Small Interfering Rnas Specific To Dock8 Sirna Dock8 2, supplied by Shanghai Genechem Ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/small interfering rnas specific to dock8 sirna-dock8-2/product/Shanghai Genechem Ltd
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elisa kits for dock8  (MyBiosource Biotechnology)
90
MyBiosource Biotechnology elisa kits for dock8
A novel compound heterozygous mutation in <t>DOCK8</t> results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.
Elisa Kits For Dock8, supplied by MyBiosource Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dock8 (nm_203447) human tagged orf clone  (OriGene)
90
OriGene dock8 (nm_203447) human tagged orf clone
A novel compound heterozygous mutation in <t>DOCK8</t> results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.
Dock8 (Nm 203447) Human Tagged Orf Clone, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


(A) Immunoblots showing the phosphoinositide-binding specificity of DOCK8. Lysates of BW5147α − β − cells expressing HA-tagged wild-type (WT) DOCK8 were used as an input for incubation with lipid vesicles containing each phosphoinositide at indicated concentrations (%). Lipid-associated fractions were analyzed by immunoblotting with anti-HA antibody. Positions of the size marker were shown on the right. (B) Schematic diagram of DOCK8 constructs used in the experiments. (C) Immunoblots showing no detectable binding to PI(4,5)P2 of a DOCK8 mutant in which the DHR-1 domain is deleted (ΔDHR-1). Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8-ΔDHR-1 (bottom) were used as an input for lipid-binding assays. (D) Immunoblots showing PI(4,5)P2 binding of recombinant GST-fusion DOCK8 DHR-1 domain (top) and GST-fusion PLCδ1 PH domain (bottom).

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Immunoblots showing the phosphoinositide-binding specificity of DOCK8. Lysates of BW5147α − β − cells expressing HA-tagged wild-type (WT) DOCK8 were used as an input for incubation with lipid vesicles containing each phosphoinositide at indicated concentrations (%). Lipid-associated fractions were analyzed by immunoblotting with anti-HA antibody. Positions of the size marker were shown on the right. (B) Schematic diagram of DOCK8 constructs used in the experiments. (C) Immunoblots showing no detectable binding to PI(4,5)P2 of a DOCK8 mutant in which the DHR-1 domain is deleted (ΔDHR-1). Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8-ΔDHR-1 (bottom) were used as an input for lipid-binding assays. (D) Immunoblots showing PI(4,5)P2 binding of recombinant GST-fusion DOCK8 DHR-1 domain (top) and GST-fusion PLCδ1 PH domain (bottom).

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Western Blot, Binding Assay, Expressing, Incubation, Marker, Construct, Mutagenesis, Recombinant

(A) Ribbon diagram of the DOCK8 DHR-1 domain. The β-sandwich core is colored in cyan; β2-β3 loop and β7-β8 insertion are in orange. Three loops on the upper surface are labeled with numbers (L1 through L3). (B) Ribbon diagram of the DOCK1 DHR-1 domain (PDB ID: 3L4C ; gray and light-yellow). (C, D, E, F) Surface charge representation of the DOCK8 DHR-1 domain (C, E) and the DOCK1 DHR-1 domain (D, F). Blue and red represent positive and negative electrostatic potential, respectively. The views in (C, D) are the same as those in (A, B); their top views are shown in (E, F), respectively. The upper surface region, β-groove, and basic pocket are indicated. (G) Ribbon diagram of the DOCK8 DHR-1 domain in the presence of 0.84 mM diC8-PI(4,5)P2 (purple) superposed with the one in the absence of PI(4,5)P2 shown in (A) (cyan). (H) Close-up view of the upper surface pocket of the DOCK8 DHR-1 domain. The boxed region in (G) is slightly tilted for the frontal view of the L1 loop region. Residues R570, K576, and R581 are highlighted by a stick model to show the different conformations.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Ribbon diagram of the DOCK8 DHR-1 domain. The β-sandwich core is colored in cyan; β2-β3 loop and β7-β8 insertion are in orange. Three loops on the upper surface are labeled with numbers (L1 through L3). (B) Ribbon diagram of the DOCK1 DHR-1 domain (PDB ID: 3L4C ; gray and light-yellow). (C, D, E, F) Surface charge representation of the DOCK8 DHR-1 domain (C, E) and the DOCK1 DHR-1 domain (D, F). Blue and red represent positive and negative electrostatic potential, respectively. The views in (C, D) are the same as those in (A, B); their top views are shown in (E, F), respectively. The upper surface region, β-groove, and basic pocket are indicated. (G) Ribbon diagram of the DOCK8 DHR-1 domain in the presence of 0.84 mM diC8-PI(4,5)P2 (purple) superposed with the one in the absence of PI(4,5)P2 shown in (A) (cyan). (H) Close-up view of the upper surface pocket of the DOCK8 DHR-1 domain. The boxed region in (G) is slightly tilted for the frontal view of the L1 loop region. Residues R570, K576, and R581 are highlighted by a stick model to show the different conformations.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Labeling

(A, B) Stereo views of the 2Fo-Fc electron density map (contoured at 0.6 σ) around L1 loop of DOCK8 DHR-1 in the absence (A), and presence (B) of 0.84 mM diC8-PI(4,5)P2 are shown.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A, B) Stereo views of the 2Fo-Fc electron density map (contoured at 0.6 σ) around L1 loop of DOCK8 DHR-1 in the absence (A), and presence (B) of 0.84 mM diC8-PI(4,5)P2 are shown.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques:

(A) Immunoblots showing the effect of point mutations on PI(4,5)P2 binding of GST-fusion DOCK8 DHR-1 domain. Mutant proteins carry alanine substitution at the indicated residue. A doubly mutant carrying K576A and R581A is designated as “KARA.” (B, C) Measurement of PI(4,5)P2 binding to DOCK8 DHR-1 by isothermal titration calorimetry. Conditions: 0.062 mM DOCK8 DHR-1 protein titrated with 2 μl aliquotes of 1 mM diC8-PI(4,5)P2 in 20 mM Tris (pH 8.0) and 16 mM NaCl at 25°C. (B) Representative titration plots for each DOCK8 DHR-1 (n = 3). Data were best fitted to acquire the stoichiometry and thermodynamic parameters. (C) Summary of the experiments. Kd: dissociation constant; ΔH: enthalpy change; TΔS: temperature (K) x entropy change; N: stoichiometry. Data were expressed as means ± SD (n = 3). (D) Immunoblots showing no detectable binding of DOCK8 KARA mutant to PI(4,5)P2. Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8 KARA (bottom) were used as an input for lipid-binding assays.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Immunoblots showing the effect of point mutations on PI(4,5)P2 binding of GST-fusion DOCK8 DHR-1 domain. Mutant proteins carry alanine substitution at the indicated residue. A doubly mutant carrying K576A and R581A is designated as “KARA.” (B, C) Measurement of PI(4,5)P2 binding to DOCK8 DHR-1 by isothermal titration calorimetry. Conditions: 0.062 mM DOCK8 DHR-1 protein titrated with 2 μl aliquotes of 1 mM diC8-PI(4,5)P2 in 20 mM Tris (pH 8.0) and 16 mM NaCl at 25°C. (B) Representative titration plots for each DOCK8 DHR-1 (n = 3). Data were best fitted to acquire the stoichiometry and thermodynamic parameters. (C) Summary of the experiments. Kd: dissociation constant; ΔH: enthalpy change; TΔS: temperature (K) x entropy change; N: stoichiometry. Data were expressed as means ± SD (n = 3). (D) Immunoblots showing no detectable binding of DOCK8 KARA mutant to PI(4,5)P2. Lysates of BW5147α − β − cells expressing HA-tagged WT DOCK8 (top) or DOCK8 KARA (bottom) were used as an input for lipid-binding assays.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Western Blot, Binding Assay, Mutagenesis, Residue, Isothermal Titration Calorimetry, Titration, Expressing

Conditions: 0.062 mM DOCK8 DHR-1 titrated with 2 μl aliquotes of 1 mM diC8-PI(3,4,5)P3 in 20 mM Tris (pH 8.0), 16 mM NaCl at 25°C. (A) Representative titration plots for WT DOCK8 DHR-1 and KARA mutant (n = 5). (B) Data were best fitted to acquire the stoichiometry and thermodynamic parameters.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: Conditions: 0.062 mM DOCK8 DHR-1 titrated with 2 μl aliquotes of 1 mM diC8-PI(3,4,5)P3 in 20 mM Tris (pH 8.0), 16 mM NaCl at 25°C. (A) Representative titration plots for WT DOCK8 DHR-1 and KARA mutant (n = 5). (B) Data were best fitted to acquire the stoichiometry and thermodynamic parameters.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Titration, Mutagenesis

(A) Images of diC8-PI(4,5)P2 docked into the upper surface pocket of the DOCK8 DHR-1 domain. PI(4,5)P2 are highlighted by a stick model. DOCK8 DHR-1 is shown in ribbon diagram (green). (B) Close-up view of the upper surface pocket in (A). Residues predicted to form the pocket and/or bind phospholipid are shown. (C) Surface charge representation of DOCK8 DHR-1 in (A) showing the electrostatic surface of the phospholipid-binding pocket. (D) Close-up view of the binding pocket in (C).

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Images of diC8-PI(4,5)P2 docked into the upper surface pocket of the DOCK8 DHR-1 domain. PI(4,5)P2 are highlighted by a stick model. DOCK8 DHR-1 is shown in ribbon diagram (green). (B) Close-up view of the upper surface pocket in (A). Residues predicted to form the pocket and/or bind phospholipid are shown. (C) Surface charge representation of DOCK8 DHR-1 in (A) showing the electrostatic surface of the phospholipid-binding pocket. (D) Close-up view of the binding pocket in (C).

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Binding Assay

Shown are the L1-loop regions of the DOCK-C subfamily: vertebrate DOCK8 (human/mouse/bird/reptile/frog/fish), mouse DOCK6 and DOCK7, and Drosophila Zizimin–related (Zir), and the DOCK-A subfamily: mouse DOCK1 and DOCK2, and Drosophila myoblast city (Mbc), aligned by the Clustal Omega software. Numbers on the top indicate the amino acid position in mouse DOCK8. Conserved basic residues are marked by red boxes. The secondary structure is indicated.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: Shown are the L1-loop regions of the DOCK-C subfamily: vertebrate DOCK8 (human/mouse/bird/reptile/frog/fish), mouse DOCK6 and DOCK7, and Drosophila Zizimin–related (Zir), and the DOCK-A subfamily: mouse DOCK1 and DOCK2, and Drosophila myoblast city (Mbc), aligned by the Clustal Omega software. Numbers on the top indicate the amino acid position in mouse DOCK8. Conserved basic residues are marked by red boxes. The secondary structure is indicated.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Software

Close-up view of the upper surface pocket of DOCK8 DHR-1 R570S mutant (in ribbon diagram) docked with diC8-PI(4,5)P2 (shown in yello stick). S570 is capable of forming a hydrogen bond with the phosphate at one position of the inositol ring.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: Close-up view of the upper surface pocket of DOCK8 DHR-1 R570S mutant (in ribbon diagram) docked with diC8-PI(4,5)P2 (shown in yello stick). S570 is capable of forming a hydrogen bond with the phosphate at one position of the inositol ring.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Mutagenesis

(A) Confocal images showing the cellular localization of HA-tagged WT and mutant DOCK8. BW5147α − β − cells stably expressing indicated proteins were analyzed by immunofluorescence using anti-HA antibody (red). Alexa Fluor 488–conjugated WGA (green) and DAPI (4′,6-diamidino-2-phenylindole, blue) were used to stain the cell surface membrane and nucleus, respectively. Scale bar: 10 μm. (B) Line scanned intensity profiles for HA and WGA fluorescence in the respective cells depicted in (A). Fluorescence intensity of HA and WGA stainings was scanned along the dotted lines in (A). The x-axis indicates arbitrary position on the line. (C) Quantification of PM localization of WT and mutant DOCK8. The intensity profiles for HA (red) and WGA (green) fluorescence from mupltiple cells were averaged (n = 48/24, 44/22, and 34/18 regions/cells for DOCK8 WT, KARA, and ΔDHR-1, respectively). Data are means ± SD. Positions of the peak intensity of WGA fluorescence were defined as the PM, and the fluorescence intensity at PM was set as 1.0 for normalization of HA and WGA fluorescence in individual cells. Distance from PM was plotted on x-axis. (D) Plasma membrane to cytoplasmic ratio of HA fluorescence for WT and mutant DOCK8 protein. The ratio of HA fluorescence intensity at the PM and cytoplasm (Cyto) in (C) was plotted for individual cells. ** P < 0.0001 by a two-tailed unpaired Mann-Whitney test. (E) Fluorescence resonance energy transfer (FRET)–based measurement of Cdc42 activity in living cells. COS-7 cells were co-transfected with a FRET-based biosensor (Raichu-Cdc42), and the pBJ-neo or the respective DOCK8 constructs. FRET imaging was performed during 26–32 h after transfection. Relative emission ratio (YFP/CFP) of the whole cell area was calculated (n = 10, 20, 17, and 14 cells from three independent experiments for control, WT, KARA, and ΔDHR-1, respectively). P -values by a two-tailed unpaired t test. Right panel: Immunoblots showing the expression of transfected DOCK8 and Raichu-Cdc42 (probed with anti-HA and anti-GFP antibodies, respectively). (F) 3D migration in collagen gels of BW5147α − β − cells expressing HA-tagged DOCK2 and FLAG-tagged WT or mutant DOCK8 (n = 232–286 cells per group from three independent experiments). Two independent clones were analyzed for KARA mutation. Each box plot indicates the median (the line in the middle), 25th and 75th percentiles (box ends), and 10th and 90th percentiles (whiskers). The number on each column indicates the average speed in μm/min. ** P < 0.0001 by a two-tailed unpaired Mann–Whitney test. Right panel: immunoblots showing the expression level of DOCK2 and DOCK8 in the cells. Actin blot is shown as a loading control; the positions for the size markers on the right.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Confocal images showing the cellular localization of HA-tagged WT and mutant DOCK8. BW5147α − β − cells stably expressing indicated proteins were analyzed by immunofluorescence using anti-HA antibody (red). Alexa Fluor 488–conjugated WGA (green) and DAPI (4′,6-diamidino-2-phenylindole, blue) were used to stain the cell surface membrane and nucleus, respectively. Scale bar: 10 μm. (B) Line scanned intensity profiles for HA and WGA fluorescence in the respective cells depicted in (A). Fluorescence intensity of HA and WGA stainings was scanned along the dotted lines in (A). The x-axis indicates arbitrary position on the line. (C) Quantification of PM localization of WT and mutant DOCK8. The intensity profiles for HA (red) and WGA (green) fluorescence from mupltiple cells were averaged (n = 48/24, 44/22, and 34/18 regions/cells for DOCK8 WT, KARA, and ΔDHR-1, respectively). Data are means ± SD. Positions of the peak intensity of WGA fluorescence were defined as the PM, and the fluorescence intensity at PM was set as 1.0 for normalization of HA and WGA fluorescence in individual cells. Distance from PM was plotted on x-axis. (D) Plasma membrane to cytoplasmic ratio of HA fluorescence for WT and mutant DOCK8 protein. The ratio of HA fluorescence intensity at the PM and cytoplasm (Cyto) in (C) was plotted for individual cells. ** P < 0.0001 by a two-tailed unpaired Mann-Whitney test. (E) Fluorescence resonance energy transfer (FRET)–based measurement of Cdc42 activity in living cells. COS-7 cells were co-transfected with a FRET-based biosensor (Raichu-Cdc42), and the pBJ-neo or the respective DOCK8 constructs. FRET imaging was performed during 26–32 h after transfection. Relative emission ratio (YFP/CFP) of the whole cell area was calculated (n = 10, 20, 17, and 14 cells from three independent experiments for control, WT, KARA, and ΔDHR-1, respectively). P -values by a two-tailed unpaired t test. Right panel: Immunoblots showing the expression of transfected DOCK8 and Raichu-Cdc42 (probed with anti-HA and anti-GFP antibodies, respectively). (F) 3D migration in collagen gels of BW5147α − β − cells expressing HA-tagged DOCK2 and FLAG-tagged WT or mutant DOCK8 (n = 232–286 cells per group from three independent experiments). Two independent clones were analyzed for KARA mutation. Each box plot indicates the median (the line in the middle), 25th and 75th percentiles (box ends), and 10th and 90th percentiles (whiskers). The number on each column indicates the average speed in μm/min. ** P < 0.0001 by a two-tailed unpaired Mann–Whitney test. Right panel: immunoblots showing the expression level of DOCK2 and DOCK8 in the cells. Actin blot is shown as a loading control; the positions for the size markers on the right.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Mutagenesis, Stable Transfection, Expressing, Immunofluorescence, Staining, Membrane, Fluorescence, Clinical Proteomics, Two Tailed Test, MANN-WHITNEY, Förster Resonance Energy Transfer, Activity Assay, Transfection, Construct, Imaging, Control, Western Blot, Migration, Clone Assay

(A) Immunoblots showing the subcellular distribution of WT and mutant DOCK8. Cytoplasmic “C” and membrane “M” fractions were prepared from BW5147α − β − cells expressing HA-tagged DOCK8 WT, KARA, or ΔDHR-1 and analyzed by immunoblotting. Input “I” (total cell lysate), C, and M fractions were loaded at 1:2:4 ratio for visualization. LAT (the linker for activation of T cells; transmembrane protein) and Cdc42 (mainly cytoplasmic) were also probed to assess the integrity of the fractions. (B) Plot showing the membrane to cytoplasmic ratio for each DOCK8. Data from densitometric analyses of immunoblots from three independent experiments.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Immunoblots showing the subcellular distribution of WT and mutant DOCK8. Cytoplasmic “C” and membrane “M” fractions were prepared from BW5147α − β − cells expressing HA-tagged DOCK8 WT, KARA, or ΔDHR-1 and analyzed by immunoblotting. Input “I” (total cell lysate), C, and M fractions were loaded at 1:2:4 ratio for visualization. LAT (the linker for activation of T cells; transmembrane protein) and Cdc42 (mainly cytoplasmic) were also probed to assess the integrity of the fractions. (B) Plot showing the membrane to cytoplasmic ratio for each DOCK8. Data from densitometric analyses of immunoblots from three independent experiments.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Western Blot, Mutagenesis, Membrane, Expressing, Activation Assay

(A) Immunoblot analysis of the expression level of DOCK8 protein in DCs derived from DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mice. Actin blot is shown as a loading control. The positions for the size markers were indicated on the right. (B) Impaired migration of DOCK8 KARA/− and DOCK8 −/− mature DCs (mDCs) in 3D collagen gels. Migration of LPS-stimulated DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs toward CCL21 source was recorded for 120 min by time-lapse video microscopy. Representative tracks of individual mDCs. (C) The migration speed, directionality, and forward migration index were compared among DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs (n = 132 (109), 127 (75), and 100 (41), respectively, from three independent experiments). For directionality and forward migration index, the cells that had migrated at 0.3 μm/min or faster were analyzed (cell numbers in the parentheses). Each box plot indicates the median (the line in the middle), 25th and 75th percentiles (box ends), and 10th and 90th percentiles (whiskers). P -values by a two-tailed unpaired Mann–Whitney test. (D) In vivo migration efficiency of DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs. DCs in a pair were mixed at 1:1 ratio, injected into footpads of C57BL/6 mice and recovered from the popliteal LNs after 48 h. Data are means ± SD for six pairs of DOCK8 WT/− and DOCK8 KARA/− DCs with data for two pairs of DOCK8 WT/− and DOCK8 −/− DCs. P -value by a two-tailed unpaired Mann–Whitney test.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) Immunoblot analysis of the expression level of DOCK8 protein in DCs derived from DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mice. Actin blot is shown as a loading control. The positions for the size markers were indicated on the right. (B) Impaired migration of DOCK8 KARA/− and DOCK8 −/− mature DCs (mDCs) in 3D collagen gels. Migration of LPS-stimulated DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs toward CCL21 source was recorded for 120 min by time-lapse video microscopy. Representative tracks of individual mDCs. (C) The migration speed, directionality, and forward migration index were compared among DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs (n = 132 (109), 127 (75), and 100 (41), respectively, from three independent experiments). For directionality and forward migration index, the cells that had migrated at 0.3 μm/min or faster were analyzed (cell numbers in the parentheses). Each box plot indicates the median (the line in the middle), 25th and 75th percentiles (box ends), and 10th and 90th percentiles (whiskers). P -values by a two-tailed unpaired Mann–Whitney test. (D) In vivo migration efficiency of DOCK8 WT/− , DOCK8 KARA/− , and DOCK8 −/− mDCs. DCs in a pair were mixed at 1:1 ratio, injected into footpads of C57BL/6 mice and recovered from the popliteal LNs after 48 h. Data are means ± SD for six pairs of DOCK8 WT/− and DOCK8 KARA/− DCs with data for two pairs of DOCK8 WT/− and DOCK8 −/− DCs. P -value by a two-tailed unpaired Mann–Whitney test.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Western Blot, Expressing, Derivative Assay, Control, Migration, Microscopy, Two Tailed Test, MANN-WHITNEY, In Vivo, Injection

(A) CCR7 mRNA expression was quantified by real time qPCR from three independent DC preparations. Data are shown in fold increase relative to the level of DOCK8 WT/− DCs (means ± SD). Neutophils were analyzed as a negative control. (B) Cell surface presentation of CCR7 was analyzed by flow cytometry with DCs from three independent preparations. (C) Representative flow cytometry plots. DCs were stained with phosphatidylethanolamine–conjugated anti-CCR7 or isotype-matched control antibody.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: (A) CCR7 mRNA expression was quantified by real time qPCR from three independent DC preparations. Data are shown in fold increase relative to the level of DOCK8 WT/− DCs (means ± SD). Neutophils were analyzed as a negative control. (B) Cell surface presentation of CCR7 was analyzed by flow cytometry with DCs from three independent preparations. (C) Representative flow cytometry plots. DCs were stained with phosphatidylethanolamine–conjugated anti-CCR7 or isotype-matched control antibody.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Expressing, Negative Control, Flow Cytometry, Staining, Control

Through the DHR-1 domain, DOCK8 is localized to a PI(4,5)P2–enriched compartment of the plasma membrane, where, upon encounter with GDP-Cdc42, the DHR-2 domain catalyzes the nucleotide exchange reaction of Cdc42 (the current work marked in a box). GTP-loaded Cdc42 and PI(4,5)P2 serve as the coincidence detection signals for activation of Wiskott-Aldrich syndrome protein, which stimulates Arp2/3 complex–mediated actin polymerization for reorganization of the actin cytoskeleton. In a similar, but distinct way, the DOCK-A subfamily member (e.g., DOCK2), which makes a signaling complex with engulfment and cell motility (ELMO) protein through the SH3 domain ( ; ), is recruited to the leading edge of migrating cells through the PI(3,4,5)P3–binding DHR-1 domain in response to chemoattractant signals. The DHR-2 domain of the DOCK-A subfamily activates Rac, which in turn acts in synergy with PI(3,4,5)P3 to activate WAVE complex. See text for details.

Journal: Life Science Alliance

Article Title: A conserved PI(4,5)P2–binding domain is critical for immune regulatory function of DOCK8

doi: 10.26508/lsa.202000873

Figure Lengend Snippet: Through the DHR-1 domain, DOCK8 is localized to a PI(4,5)P2–enriched compartment of the plasma membrane, where, upon encounter with GDP-Cdc42, the DHR-2 domain catalyzes the nucleotide exchange reaction of Cdc42 (the current work marked in a box). GTP-loaded Cdc42 and PI(4,5)P2 serve as the coincidence detection signals for activation of Wiskott-Aldrich syndrome protein, which stimulates Arp2/3 complex–mediated actin polymerization for reorganization of the actin cytoskeleton. In a similar, but distinct way, the DOCK-A subfamily member (e.g., DOCK2), which makes a signaling complex with engulfment and cell motility (ELMO) protein through the SH3 domain ( ; ), is recruited to the leading edge of migrating cells through the PI(3,4,5)P3–binding DHR-1 domain in response to chemoattractant signals. The DHR-2 domain of the DOCK-A subfamily activates Rac, which in turn acts in synergy with PI(3,4,5)P3 to activate WAVE complex. See text for details.

Article Snippet: Immunoblotting was performed with following antibodies: rat monoclonal antibody for HA (1:2,000 dilution, 3F10; Roche), anti-GST antibody (1:500, 013-21851; Wako), rabbit anti-GFP antibody (1:1,000, A11122; Invitrogen), mouse anti-Cdc42 antibody (1:1,000, 05-542; Millipore), rabbit anti-LAT antibody (1:1,000 06-807; Millipore), HRP-conjugated rabbit anti-FLAG (DDDDK-tag) antibody (1:2,000, PM020-7; MBL), custom-made rabbit anti-DOCK8 antibody (1:1,000; ), goat anti-actin (1:1,000, sc-1616; Santa Cruz), and corresponding species-specific HRP-conjugated anti-IgG antibodies (1:2,000; all from Santa Cruz).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Binding Assay

Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release HMGB1 as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release HMGB1 as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Injection, Activation Assay, Expressing, Inhibition

Fig. 1. Celastrol (CEL) induces ICD and down-regulates PD-L1 expression in melanoma in vitro and in vivo. (A) Chemical structure of CEL. (B) CEL triggered autophagy in melanoma cells. Mouse B16F10 or BPD6 melanoma cells were incubated with CEL (0.1–8 μM) for 12 h and then lysed for Western blot analysis of LC3B, whose subunit transition from LC3B I to LC3B II is a marker for activated autophagy. (C) Immunofluorescent imaging of CRT, HMGB1, and PD-L1 in mouse (B16F10 and BPD6) and human (M10 and A375) melanoma cells after treated with CEL at 1 μM; The treatment time was 4 h for CRT and 24 h for HMGB1 and PD-L1 detection. Cell nuclei were stained with DAPI. Scale bar indicates 20 μm. Each value was quantified in 5 randomly selected fields. Each sample was repeated 3 times. (D) Flow cytometry analysis of CRT+ melanoma cells after treated with CEL or positive control, doxorubicin (DOX) and mitoxantrone (MIT), for 4 h at their IC50. (E) The released HMGB1 in the cell culture medium 24 h after incubation with CEL, DOX or MIT at their IC50 doses (n = 4). (F) RT-PCR analysis of the PD-L1 mRNA levels in B16F10 and BPD6 cells after incubation with CEL, DOX or MIT at IC50 for 24 h (n = 6). (G) Western blot analysis of PD-L1 expression in tumors at 48 h after i.t. injection with CEL (0.15 mg/kg), DOX (0.01 mg/kg) or MIT (0.5 mg/kg) or i.p. injection with αPD-L1 (5 mg/kg). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 1. Celastrol (CEL) induces ICD and down-regulates PD-L1 expression in melanoma in vitro and in vivo. (A) Chemical structure of CEL. (B) CEL triggered autophagy in melanoma cells. Mouse B16F10 or BPD6 melanoma cells were incubated with CEL (0.1–8 μM) for 12 h and then lysed for Western blot analysis of LC3B, whose subunit transition from LC3B I to LC3B II is a marker for activated autophagy. (C) Immunofluorescent imaging of CRT, HMGB1, and PD-L1 in mouse (B16F10 and BPD6) and human (M10 and A375) melanoma cells after treated with CEL at 1 μM; The treatment time was 4 h for CRT and 24 h for HMGB1 and PD-L1 detection. Cell nuclei were stained with DAPI. Scale bar indicates 20 μm. Each value was quantified in 5 randomly selected fields. Each sample was repeated 3 times. (D) Flow cytometry analysis of CRT+ melanoma cells after treated with CEL or positive control, doxorubicin (DOX) and mitoxantrone (MIT), for 4 h at their IC50. (E) The released HMGB1 in the cell culture medium 24 h after incubation with CEL, DOX or MIT at their IC50 doses (n = 4). (F) RT-PCR analysis of the PD-L1 mRNA levels in B16F10 and BPD6 cells after incubation with CEL, DOX or MIT at IC50 for 24 h (n = 6). (G) Western blot analysis of PD-L1 expression in tumors at 48 h after i.t. injection with CEL (0.15 mg/kg), DOX (0.01 mg/kg) or MIT (0.5 mg/kg) or i.p. injection with αPD-L1 (5 mg/kg). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Expressing, In Vitro, In Vivo, Incubation, Western Blot, Marker, Imaging, Staining, Flow Cytometry, Positive Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Injection

Fig. 2. Characterization of celastrol nanoemulsion (CEL NE) and its ICD-induction and PD-L1 downregulation on melanoma. (A) The size distribution of CEL NE characterized by dynamic laser light scattering and transmission electron microscopy (insets). (B) The dose-dependent curve of CRT+-population of B16F10 cells incubated with free CEL or CEL NE for 4 h. The percentage of CRT+ cells was quantified as an average of 5 randomly selected fields. Each sample was repeated 3 times; see Supplementary Fig. S4 for the curves of other cells. (C–F) Analysis of in vivo ICD-induction and PD-L1 downregulation. The mice bearing B16F10 tumors received a single i.t. injection of CEL NE or free CEL (0.15 mg/kg) (n = 4) and were sacrificed after 48 h for analysis: (C) Representative immunofluorescent images of CRT, HMGB1 and activated DC markers (CD86, CD11c, and MHC II) in B16F10 tumors after i.t. injection of CEL NE or free CEL. Each value was quantified as an average of 5 randomly selected fields. Scale bar indicates 300 μm. (D) Statistic analysis of co-stimulatory markers, CD80 and CD86, in the draining lymph nodes by flow cytometry (n = 4). (E) Flow cytometry quantitation of PD-L1+ melanoma cell percentages (PD-L1+MART+) in the tumors. (F) RT-PCR analysis of PD-L1 mRNA levels in the tumors. (G) Western blot analysis of dose-dependent downregulation of NF-κB and PD-L1 in the tumors. The mice with tumors as above were i.t. injected with the indicated doses of CEL NE and analyzed after 48 h (n = 4). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 2. Characterization of celastrol nanoemulsion (CEL NE) and its ICD-induction and PD-L1 downregulation on melanoma. (A) The size distribution of CEL NE characterized by dynamic laser light scattering and transmission electron microscopy (insets). (B) The dose-dependent curve of CRT+-population of B16F10 cells incubated with free CEL or CEL NE for 4 h. The percentage of CRT+ cells was quantified as an average of 5 randomly selected fields. Each sample was repeated 3 times; see Supplementary Fig. S4 for the curves of other cells. (C–F) Analysis of in vivo ICD-induction and PD-L1 downregulation. The mice bearing B16F10 tumors received a single i.t. injection of CEL NE or free CEL (0.15 mg/kg) (n = 4) and were sacrificed after 48 h for analysis: (C) Representative immunofluorescent images of CRT, HMGB1 and activated DC markers (CD86, CD11c, and MHC II) in B16F10 tumors after i.t. injection of CEL NE or free CEL. Each value was quantified as an average of 5 randomly selected fields. Scale bar indicates 300 μm. (D) Statistic analysis of co-stimulatory markers, CD80 and CD86, in the draining lymph nodes by flow cytometry (n = 4). (E) Flow cytometry quantitation of PD-L1+ melanoma cell percentages (PD-L1+MART+) in the tumors. (F) RT-PCR analysis of PD-L1 mRNA levels in the tumors. (G) Western blot analysis of dose-dependent downregulation of NF-κB and PD-L1 in the tumors. The mice with tumors as above were i.t. injected with the indicated doses of CEL NE and analyzed after 48 h (n = 4). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Transmission Assay, Electron Microscopy, Incubation, In Vivo, Injection, Flow Cytometry, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

STAT3 phosphorylation analysis after stimulation. ( a ) Western blot analysis of whole cell lysates of PBMCs, unstimulated or 20 min. stimulated with 200 ng/ml IL6 or IL10. Expression of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 (STAT3) of the two affected siblings and a healthy control was assessed; Actin as loading control. ( b ) Representative flow cytometric analysis showing diminished Y705-STAT3 phosphorylation after 20 min. stimulation with 200 ng/ml IL6 (solid line) versus unremarkable results after stimulation with 20 ng/ml IL10 (dotted line) and 10 ng/ml IL21 (dashed line) in lymphocytes of patient II.2 compared to unremarkable results in patient II.3 and a healthy control; filled gray area: unstimulated lymphocytes. ( c ) Flow cytometric analysis showing Y705-STAT3 phosphorylation after 20 min. stimulation with 20 ng/ml IL6 (solid line) or IL10 (dotted line) and 10 ng/ml IL21 (dashed line) comparable to healthy control in lymphocytes of one (representative of four) DOCK8-HIES patient. ( d ) Restored STAT3 phosphorylation after IL6 stimulation (solid line) in patient II.2 15 months after HSCT compared to unstimulated (filled gray area) and IL10-stimulated (dotted line) lymphocytes.

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: STAT3 phosphorylation analysis after stimulation. ( a ) Western blot analysis of whole cell lysates of PBMCs, unstimulated or 20 min. stimulated with 200 ng/ml IL6 or IL10. Expression of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 (STAT3) of the two affected siblings and a healthy control was assessed; Actin as loading control. ( b ) Representative flow cytometric analysis showing diminished Y705-STAT3 phosphorylation after 20 min. stimulation with 200 ng/ml IL6 (solid line) versus unremarkable results after stimulation with 20 ng/ml IL10 (dotted line) and 10 ng/ml IL21 (dashed line) in lymphocytes of patient II.2 compared to unremarkable results in patient II.3 and a healthy control; filled gray area: unstimulated lymphocytes. ( c ) Flow cytometric analysis showing Y705-STAT3 phosphorylation after 20 min. stimulation with 20 ng/ml IL6 (solid line) or IL10 (dotted line) and 10 ng/ml IL21 (dashed line) comparable to healthy control in lymphocytes of one (representative of four) DOCK8-HIES patient. ( d ) Restored STAT3 phosphorylation after IL6 stimulation (solid line) in patient II.2 15 months after HSCT compared to unstimulated (filled gray area) and IL10-stimulated (dotted line) lymphocytes.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Genetic analysis of DOCK8 . ( a ) T cell blast cDNA chromatograms show wildtype sequence in a healthy control, double peaks in patient II.2 and altered sequence in patient II.3. Both patients’ gDNA is homozygous for alteration c.4626 + 76 A > G; vertical black lines: 3′ junction of exon 36; black letters: wildtype; red letters: altered sequence. ( b ) Schematic model of affected region in DOCK8 gDNA and transcripts showing exon extension (dotted line) due to the novel splice site (*) introduced at c.4626 + 76 A > G; filled boxes: exons; horizontal line: intronic region. ( c ) Quantification of wildtype and altered transcripts in T cell blasts by ddPCR indicating percentages of wildtype (wt) of total DOCK8 transcripts in patient II.2, patient II.3 and healthy controls (HC). ( d ) ddPCR analysis of healthy controls (homozygous wt) and healthy carriers of a c.3120 + 1 G > T DOCK8 alteration resulting in exon 25 skipping (heterozygous). ( e ) Sashimi plot of RNA sequencing data based on GTEx samples , showing exon 32 skipping as a rare event; read counts accumulated over all samples. ( f ) Schematic model of wildtype and mutated minigene vectors. Sequence tags (PT1/PT2) flanked the minigene sequence to differentiate minigene transcripts from endogenous DOCK8 transcripts; filled boxes: exons; dotted line: exon extension; horizontal line: intronic regions; *: novel splice site. ( g ) The altered or physiologic transcription products of the minigene vectors were differentiated by size. Agarose gel with canonical splice site usage (378 nucleotide transcript) in cDNA of control PBMCs transfected with wildtype (Mini wt) and usage of the novel splice site (453 nucleotide transcript) in cDNA of PBMCs transfected with the mutated minigene vector (Mini mut); GFP- and mock-transfected as negative controls.

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: Genetic analysis of DOCK8 . ( a ) T cell blast cDNA chromatograms show wildtype sequence in a healthy control, double peaks in patient II.2 and altered sequence in patient II.3. Both patients’ gDNA is homozygous for alteration c.4626 + 76 A > G; vertical black lines: 3′ junction of exon 36; black letters: wildtype; red letters: altered sequence. ( b ) Schematic model of affected region in DOCK8 gDNA and transcripts showing exon extension (dotted line) due to the novel splice site (*) introduced at c.4626 + 76 A > G; filled boxes: exons; horizontal line: intronic region. ( c ) Quantification of wildtype and altered transcripts in T cell blasts by ddPCR indicating percentages of wildtype (wt) of total DOCK8 transcripts in patient II.2, patient II.3 and healthy controls (HC). ( d ) ddPCR analysis of healthy controls (homozygous wt) and healthy carriers of a c.3120 + 1 G > T DOCK8 alteration resulting in exon 25 skipping (heterozygous). ( e ) Sashimi plot of RNA sequencing data based on GTEx samples , showing exon 32 skipping as a rare event; read counts accumulated over all samples. ( f ) Schematic model of wildtype and mutated minigene vectors. Sequence tags (PT1/PT2) flanked the minigene sequence to differentiate minigene transcripts from endogenous DOCK8 transcripts; filled boxes: exons; dotted line: exon extension; horizontal line: intronic regions; *: novel splice site. ( g ) The altered or physiologic transcription products of the minigene vectors were differentiated by size. Agarose gel with canonical splice site usage (378 nucleotide transcript) in cDNA of control PBMCs transfected with wildtype (Mini wt) and usage of the novel splice site (453 nucleotide transcript) in cDNA of PBMCs transfected with the mutated minigene vector (Mini mut); GFP- and mock-transfected as negative controls.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Sequencing, Control, RNA Sequencing, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation

DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control. Full-length western blots are provided in the Supplementary Appendix (Supplementary Fig. ). ( b ) Flow cytometry of patient II.2 showed DOCK8 expression in majority of NK cells and T cells but no DOCK8 expression in B cells. All cell subsets of patient II.3 lack DOCK8 expression. Gray area: unstained; dashed line: isotype control; solid line: DOCK8 staining. ( c ) T cell subsets defined by naïve T cells (CCR7 + CD45RA + ), central memory T cells (CCR7 + CD45RA − ), effector memory T cells (CCR7 − CD45RA − ) and T EMRA cells (CCR7 − CD45RA + ) showed no DOCK8 expression in T EMRA and naïve T cells and DOCK8 expression in majority of central and effector memory T cells of patient II.2 compared to DOCK8 expression in all T cell subsets of a healthy control. ( d ) Sequencing of cDNA reveals double peaks in chromatograms of T and NK cells of patient II.2 indicating wildtype (black letters) and altered (red letters) transcripts. cDNA chromatogram of B cells shows only single peaks indicating altered transcripts.

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control. Full-length western blots are provided in the Supplementary Appendix (Supplementary Fig. ). ( b ) Flow cytometry of patient II.2 showed DOCK8 expression in majority of NK cells and T cells but no DOCK8 expression in B cells. All cell subsets of patient II.3 lack DOCK8 expression. Gray area: unstained; dashed line: isotype control; solid line: DOCK8 staining. ( c ) T cell subsets defined by naïve T cells (CCR7 + CD45RA + ), central memory T cells (CCR7 + CD45RA − ), effector memory T cells (CCR7 − CD45RA − ) and T EMRA cells (CCR7 − CD45RA + ) showed no DOCK8 expression in T EMRA and naïve T cells and DOCK8 expression in majority of central and effector memory T cells of patient II.2 compared to DOCK8 expression in all T cell subsets of a healthy control. ( d ) Sequencing of cDNA reveals double peaks in chromatograms of T and NK cells of patient II.2 indicating wildtype (black letters) and altered (red letters) transcripts. cDNA chromatogram of B cells shows only single peaks indicating altered transcripts.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Staining, Sequencing

Analysis of somatic alterations in DOCK8 . ( a ) Gating strategy to sort lymphocyte subsets according to their DOCK8 expression. PBMCs of patient II.2 were gated for lymphocytes and then DOCK8-negative cells into B cells (DOCK8 − CD19 + ) and non-B cells (DOCK8 − CD19 − ), and DOCK8-positive cells into T cells (DOCK8 + CD19 − CD3 + ) and NK cells (DOCK8 + CD19 − CD56 + ). ( b ) gDNA sequence of sorted cells of patient II.2 had a homozygous peak for the c.4626 + 76 A > G alteration (red letter) in T and B cells and a double peak with altered (red letter) and wildtype (black letter) sequence in NK cells. ( c ) gDNA sequence of unfixed and unpermeabilized PBMCs of patient II.2 sorted according to the lymphocyte subsets CD4 + and CD8 + T cells showing double peaks with altered (red letter) and wildtype (black letter) sequence in CD4 + T cells at positions c.4626 + 76 and c.4626 + 77 and at position c.4626 + 80 in CD8 + T cells.

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: Analysis of somatic alterations in DOCK8 . ( a ) Gating strategy to sort lymphocyte subsets according to their DOCK8 expression. PBMCs of patient II.2 were gated for lymphocytes and then DOCK8-negative cells into B cells (DOCK8 − CD19 + ) and non-B cells (DOCK8 − CD19 − ), and DOCK8-positive cells into T cells (DOCK8 + CD19 − CD3 + ) and NK cells (DOCK8 + CD19 − CD56 + ). ( b ) gDNA sequence of sorted cells of patient II.2 had a homozygous peak for the c.4626 + 76 A > G alteration (red letter) in T and B cells and a double peak with altered (red letter) and wildtype (black letter) sequence in NK cells. ( c ) gDNA sequence of unfixed and unpermeabilized PBMCs of patient II.2 sorted according to the lymphocyte subsets CD4 + and CD8 + T cells showing double peaks with altered (red letter) and wildtype (black letter) sequence in CD4 + T cells at positions c.4626 + 76 and c.4626 + 77 and at position c.4626 + 80 in CD8 + T cells.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Expressing, Sequencing

A novel compound heterozygous mutation in DOCK8 results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: A novel compound heterozygous mutation in DOCK8 results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Mutagenesis, Expressing, Sequencing, Control, Hybridization, Transformation Assay

Improvement of T cell proliferation over time, somatic reversion of DOCK8 in T cells and hypomorphic function of the truncated DOCK8 protein. Proliferation of PHA-stimulated, CFSE-labeled PBMCs of the patient at (A) 10 and (B) 15 years of age, and a healthy control. Depicted are percentages of CFSE low cells gated on CD3 + CD4 + or CD4 − or CD8 + T cells. (C) Sanger sequence trace showing somatic reversion of the single nucleotide duplication (c.6019dupT) resulting in expression of about 60% wild-type DOCK8 transcripts in the patient's CD3 + T cells. (D) “T” nucleotide phosphorescence ratios obtained by pyrosequencing DOCK8 of primary CD3 + CD4 + and CD3 + CD8 + T cells, primary CD19 + B cells and the EBV B cell line of the patient. (T/± 1 or 2) depicts the signal ratio of c.6018-19T to nucleotides 1 and 2 positions up and downstream. The PCR templates and pyrosequencing reactions were performed in triplicate. Each symbol represents the mean of the three ratio measurements at respective nucleotide positions. The bar represents the mean “T” nucleotide phosphorescence ratio of all 4 different nucleotide ratios in indicated cell populations. (E) Sanger sequence trace showing expression of solely mutated DOCK8 transcripts in EBV-transformed B cells of the patient. The duplicated T-nucleotide is indicated by the arrow and #. (F) Migration of EBV-transformed B cells of 5 different healthy controls (each symbol represents the mean of 3 independent experiments for each of the healthy control samples), the patient and a patient with a complete loss-of-function mutation in DOCK8 (DOCK8 null ). The bar of the healthy control samples represents the mean of the mean of each of the 5 healthy control samples. The bar for each of the patient samples represents mean and standard deviation of 3 independent experiments for each sample.

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: Improvement of T cell proliferation over time, somatic reversion of DOCK8 in T cells and hypomorphic function of the truncated DOCK8 protein. Proliferation of PHA-stimulated, CFSE-labeled PBMCs of the patient at (A) 10 and (B) 15 years of age, and a healthy control. Depicted are percentages of CFSE low cells gated on CD3 + CD4 + or CD4 − or CD8 + T cells. (C) Sanger sequence trace showing somatic reversion of the single nucleotide duplication (c.6019dupT) resulting in expression of about 60% wild-type DOCK8 transcripts in the patient's CD3 + T cells. (D) “T” nucleotide phosphorescence ratios obtained by pyrosequencing DOCK8 of primary CD3 + CD4 + and CD3 + CD8 + T cells, primary CD19 + B cells and the EBV B cell line of the patient. (T/± 1 or 2) depicts the signal ratio of c.6018-19T to nucleotides 1 and 2 positions up and downstream. The PCR templates and pyrosequencing reactions were performed in triplicate. Each symbol represents the mean of the three ratio measurements at respective nucleotide positions. The bar represents the mean “T” nucleotide phosphorescence ratio of all 4 different nucleotide ratios in indicated cell populations. (E) Sanger sequence trace showing expression of solely mutated DOCK8 transcripts in EBV-transformed B cells of the patient. The duplicated T-nucleotide is indicated by the arrow and #. (F) Migration of EBV-transformed B cells of 5 different healthy controls (each symbol represents the mean of 3 independent experiments for each of the healthy control samples), the patient and a patient with a complete loss-of-function mutation in DOCK8 (DOCK8 null ). The bar of the healthy control samples represents the mean of the mean of each of the 5 healthy control samples. The bar for each of the patient samples represents mean and standard deviation of 3 independent experiments for each sample.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Labeling, Control, Sequencing, Expressing, Transformation Assay, Migration, Mutagenesis, Standard Deviation