Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: CD38 expression and activity increase at late stages of ZIKV infection and correlate with NAD + decline in the brain (A) Total NADase activity measured in the brains of ZIKV-infected and mock-injected mice over the course of infection, showing significant increases from 18 dpi onward ( n ≥ 7 mice per group). (B) Overlay of NADase activity (red line) and NAD + levels (gray line), both relative to mock controls (dashed line), showing an inverse temporal association. (C–H) Relative mRNA expression of Sarm1 , Cd157 , and Cd38 , respectively, in the brains of ZIKV-infected and control mice ( n ≥ 4 mice per group). (D, F, H) Overlays of mRNA expression profiles of Sarm1 (D), Cd157 (F), and Cd38 (H) with NAD + levels (gray line) and ZIKV genomic RNA (yellow line), indicating that Cd38 induction temporally coincides with NAD + decline, while Sarm1 and Cd157 do not. (I) Linear regression shows a positive correlation between total NADase activity and Cd38 mRNA expression ( n = 50). (J) CD38-dependent NADase activity, calculated as the fraction inhibited by the specific CD38 inhibitor 78c ( n ≥ 6 mice per group). (K) CD38-independent NADase activity, which remains low and unchanged during infection ( n ≥ 6 mice per group). (L) On the right, representative Western blot of CD38 protein expression in brain extracts at 24 dpi, with α-tubulin as loading control (representative bands from the same experiment shown in the full blot in ). On the left, quantification of the Western blot bands’ intensities (CD38/α-tubulin) relative to mock ( n ≥ 4 mice per group). Data in panels A, C, E, G, J, K, and L are presented as mean ± SD; panels B, D, F, and H as mean ± SEM (shaded area). Statistical significance was determined by unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Activity Assay, Infection, Injection, Control, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: CD38 inhibition prevents NAD + depletion in the brains of ZIKV-infected mice (A) Schematic representation of the experimental design. Neonatal mice were subcutaneously infected with ZIKV at postnatal day 3 (P3). At 21 days post-infection (dpi), animals received a unilateral intracerebroventricular (i.c.v.) injection of the CD38-blocking antibody Ab68 (5.76 μg) or vehicle (Veh), and brains were collected at 24 dpi for analysis. (B) NAD + hydrolase activity in brain tissue. (C) Quantification of total NAD + levels in brain tissue. Data are presented as mean ± SD. Statistical analyses were performed using an unpaired Student’s t test ( n ≥ 7 mice per group). ∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Inhibition, Infection, Injection, Blocking Assay, Activity Assay

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: NAMPT is induced in the brain of ZIKV-infected mice (A) Relative mRNA expression of Nampt in the brains of ZIKV-infected and mock-injected mice across time points post-infection ( n ≥ 4 mice per group). (B) Overlay of Nampt mRNA expression (blue line), total NADase activity (red line), NAD + levels (gray line), and ZIKV genomic RNA (yellow line), all relative to mock controls (dashed line). (C–F) Correlation analyses between Nampt mRNA expression and (C) ZIKV genomic RNA, (D) Parp12 , (E) Parp10 , and (F) Cd38 mRNA expression. Nampt shows a strong correlation with viral load and early-induced Parps , but a weak correlation with Cd38 . (G) Representative Western blot of NAMPT protein expression in the brains of ZIKV-infected and mock-injected mice at 24 days post-infection (dpi; representative bands from the same experiment shown in the full blot in ).. HPRT was used as a loading control. The right panel shows quantification of NAMPT protein levels (NAMPT/HPRT ratio), normalized to mock controls ( n ≥ 4 mice per group). Data in panels A and G are presented as mean ± SD; panel B as mean ± SEM (shaded area). Correlations were assessed by linear regression analysis. Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Infection, Expressing, Injection, Activity Assay, Western Blot, Control, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: Proinflammatory cytokine expression precedes CD38 induction in the brains of ZIKV-infected mice (A, C, E) Relative mRNA expression of Il6 and Tnf (linear scale), and Ccl5/Rantes (Log 10 -transformed) in the brains of ZIKV-infected and mock-injected mice across time points post-infection ( n ≥ 3 mice per group). All three inflammatory mediators were significantly upregulated during the early and mid-stages of infection. (B, D, F) Overlay of Il6 (B), Tnf (D), and Rantes – log 10 scale (F) mRNA expression profiles (purple line) with Cd38 mRNA (red line), NAD + levels (gray line), and ZIKV genomic RNA (yellow line), all relative to mock controls (dashed line). (G) Brain IL-6 protein levels determined by ELISA ( n ≥ 5 mice per group). The temporal pattern shows that cytokine and chemokine induction precede Cd38 expression, suggesting that neuroinflammation may contribute to the upregulation of CD38. Data in panels A, C, and E are presented as mean ± SD; panels B, D, and F as mean ± SEM (shaded area). Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Infection, Transformation Assay, Injection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: Infiltrating immune cells contribute to increased CD38 expression in the brains of ZIKV-infected mice (A) On the left, representative dot plots show the gating strategy used to identify CD45 lo CD11b + (putative resting microglia), CD45 hi CD11b + (infiltrating myeloid cells), and CD45 + CD11b − (lymphoid cells) populations. On the right, graphs showing the frequency of each population in ZIKV-infected and mock-injected mice ( n ≥ 5 mice per group). (B) Representative dot plots and quantification of CD11b + TMEM119 + , CD11b + TMEM119 + , and CD11b + TMEM119 - populations in the brains of infected and control mice ( n ≥ 6 mice per group), with the respective graphs showing the frequency of each population. (C–E) Representative histograms and quantification of CD38 expression (median fluorescence intensity, MFI) in CD11b + TMEM119 + (C) CD11b − TMEM119 + (D), and CD11b + TMEM119 - (E) populations. (F and G) Gating and quantification of CD3 + T cells (F) and corresponding CD38 expression (G). (H and I) Gating and quantification of CD19 + B cells (H) and corresponding CD38 expression (I). All graphs represent mean ± SD. Gating was based on negative controls; histogram quantification was performed using MFI, and curves were normalized to unit area. Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Infection, Injection, Control, Fluorescence, MANN-WHITNEY

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A–C) Flow cytometry dot plots showing gating of peripheral blood B cell subsets. (D and E) CD45 phosphatase activity (D) and panCD45 surface expression (E) in human MBC and ASC subpopulations (red) compared to naive B cells (blue). Numbers in the histograms represent MFI ratio relative to naive B cells. (F and G) Graphs show pCAP-SP1 (F) and panCD45 surface expression (G) MFI values relative to naive B cells (n= 12). (H) Human BM CD19 − CD38 hi CD138 + (left panel) and CD19 + CD138 +/− CD38 + (right panel) previously shown to contain LLPCs and SLPCs, respectively. (I) CD45 phosphatase activity (left panel), CD45 surface expression (middle panel), and BLIMP1 expression (right panel) in LLPCs (red) and SLPCs (blue) relative to mature BM B cells (CD19 + SSC lo CD45 hi ) obtained from the same donor (gray). (J) Graphs show fold change in MFI of CD45 phosphatase activity (left panel) and CD45 surface expression (right panel) from human BM PCs relative to mature BM B cells from the same healthy donor (n = 4). Related to . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Activity Assay, Expressing

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry plots show gated CD38 hi CD138 hi ASCs (upper panel) and CD45 activity versus CD138 expression (lower panel) in the presence of Th signals and CD45 inhibitor as indicated. (B) Graph shows % CD38 hi CD138 hi ASC B cells (n= 8). (C) Histograms show the CD45-dependent regulation of ASC-associated markers and transcription factors. CTR+VEH (gray), CD40L+VEH (blue), CD40L+CD45 inhibitor (red). (D) Human peripheral B cells differentiated toward ASCs in the presence of CTR siRNA (upper panels) or CD45 siRNA (lower panels). (E) Graph shows % IRF4 + BLIMP1 + ASCs upon delivery of CTR or CD45 siRNA (n = 5). (F) Flow cytometry histograms show expression of CD45 activity (upper left panel), CD45 surface expression (upper right panel), BLIMP1 (lower left panel), and IRF4 (lower right panel) in purified B cells differentiated toward ASCs in the presence of CTR siRNA (gray) or CD45 siRNA (blue). Related to . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Activity Assay, Expressing, Purification

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Representative dot plots showing LN GC B cells (B220 + GL7 + CD38 − ) and high- and low-affinity HA-reactive cells; HA binding versus IgG expression is shown. (B) Histograms show CD45 activity (left) and IRF4 expression (right) in GC low- (blue-filled histograms) and high-affinity (red) B cells. (C) BM PCs gated as Blimp1 + CD138 + cells (green) overlayed on histograms of low- (blue) and high-affinity (red) GC B cells showing CD45 activity (left) and Blimp1 (middle) and CD138 (right) expression levels. (D) Dot plots show BM PC gate from forward and side scatter (F/SSC)-gated cells (left) and from B220 + SSC lo -gated cells (right). (E) PCs were backgated on dot plots depicting CD45 activity and Blimp1 (left) and CD45 activity and IRF4 (right); PCs are depicted as red dots, BM B220 + B cells are represented in blue. (F) PCs (red) and B220 + B cells (blue) were analyzed for CD45 activity (left) and HA binding (right). (G and H) Graphs show MFI of CD45 activity of low-affinity (LoAff) and high-affinity (HiAff) GC B cells and BM PCs relative to B220 + B cells within the same sample (LN or BM). (I) Graph shows MFI ratio of HA normalized to IgG. Data are representative of two experiments including seven immunized mice in addition to immunization and staining controls. Related to . *p < 0.05, **p < 0.01.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Binding Assay, Expressing, Activity Assay, Staining

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry histogram showing surface staining of Galectin-1 in naive (gray), memory (blue), and PCs (red) in human peripheral blood B cells (left panel). Isotype control is shown by gray-dotted histogram. Graph shows fold increase in surface Galectin-1 staining (MFI) relative to naive B cells (n = 4). Contour plot depicts CD45 activity versus Galectin-1 surface staining in B cells (blue) and PCs (CD138 hi CD38 hi ) (red) backgated to CD45 activity hi Galectin-1 hi cells (right panel). (B) Histograms show CD45 activity (left panel), Galectin-1 staining (middle panel), and MEM-55 staining (right panel) in CTR- or NA-treated B cells. Graphs show fold increase in MFI of CD45 phosphatase activity (left lower panel) and Galectin-1 staining (right lower panel) relative to CTR-treated B cells (n = 6). (C) Upper panels: Dot plots show expression of IRF4 and BLIMP1 in naive B cells and MBCs differentiated toward ASCs in the presence of 10 and 100 µM OTX008 or VEH. Graph shows % IRF4 + BLIMP1 + B cells in MBC cultures. Lower panels: Galectin-1 surface expression (left panel) and CD45 phosphatase activity (right panel) in VEH-treated (blue histogram) and OTX-treated (blue dotted histogram) MBCs. Graphs show MFI values of Galectin-1 staining (left) and CD45 activity (right) (n = 5). (D) Flow cytometry dot plots show CD45 activity versus Galectin-1 surface staining in the presence of medium or rhGAL-1 (left panels). Histogram shows CD45 phosphatase activity of Galectin-1 hi B cells with rhGAL1 (blue) and total B cells (gray) (middle panel). Graph shows MFI values of CD45 activity in total B cells (gray histogram) and GAL-1 hi (blue open histogram) B cells (right panel) (n = 6). (E) Representative localization of Galectin-1 relative to pSyk, pCAP-SP1, and CD45 in human B cells. The panels (left) present signals from the individual fluorescence detectors, and the center image is a merge of all four channels. The graph (right) shows the average (z axis) fluorescence intensity for each (x axis) pixel number in the indicated area (below graph and dotted area on center figure). (F) CoIP of CD45 of B cell lysates and immunoblotted with anti-CD45 (left) or anti-Galectin-1 (right). (G) Flow cytometry histogram showing CD45 phosphatase activity in gated live Raji B cells with CRISPR-Cas9 knockdown of CD45 (blue), Galectin-1 (green), and wild-type (red). Related to and . *p < 0.05, **p < 0.01.

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Flow Cytometry, Staining, Control, Activity Assay, Expressing, Fluorescence, CRISPR, Knockdown

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FITC Rat monoclonal anti-mouse CD38 (clone 90.4) , Miltenyi Biotec , Cat # 130–102-514; RRID:AB_2657875.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: Structure and in vitro characterization of AJ206. A) Structure of bicyclic peptide AJ206 having NOTA as bifunctional chelator for 68 Ga‐labeling B) Surface plasmon resonance (SPR) analysis showing affinity of AJ206 for CD38 using recombinant human CD38 protein; data is represented as mean ± SEM ( n = 2).

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: In Vitro, Labeling, SPR Assay, Recombinant

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: In vitro specificity of [ 68 Ga]Ga‐AJ206 for CD38. A) [ 68 Ga]Ga‐AJ206 binding (percent incubated activity, %IA) to different MM cells. Cells were incubated with 1 µCi [ 68 Ga]Ga‐AJ206 at 4 °C for 1 h. [ 68 Ga]Ga‐AJ206 uptake is CD38 expression dependent, and co‐incubation with 2 × 10 −6 m of nonradioactive AJ206 (blocking dose) significantly reduced radiotracer uptake confirming CD38 specificity. B) Flow cytometry analysis of CD38 surface expression in MM cells. C) CD38 receptor density in MM cells measured by quantibrite assay. D) Representative western blot of total CD38 protein expression (bottom panel). Densiometric analysis of western blot preformed using ImageJ software and band intensities represented as a ratio of CD38 protein to GAPDH control (top panel). E) Correlation of [ 68 Ga]Ga‐AJ206 uptake with surface CD38 receptor density; data in panels A, C, and E are represented as mean ± SD ( n = 3‐4). ns, P ≥ 0.05; ****, P ≤ 0.0001 is by unpaired Student's t test. Simple linear regression and Pearson coefficient were used in E.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: In Vitro, Binding Assay, Incubation, Activity Assay, Expressing, Blocking Assay, Flow Cytometry, Western Blot, Software, Control

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: In vivo specificity of [ 68 Ga]Ga‐AJ206 for CD38 in NSG mice with MM tumor xenografts. A) Whole‐body PET/CT images of different human MM xenografts at 60 min after the injection of radiotracer. Mice were injected with ≈ 7.4 MBq (≈200 µCi) [ 68 Ga]Ga‐AJ206. B) IHC staining for CD38 expression in MM xenografts. C) [ 68 Ga]Ga‐AJ206 uptake quantification (%ID g −1 ) in different MM tumors by ex vivo biodistribution at 60 min after injection. D) PET/CT images of MM1S tumor xenograft‐bearing mice with [ 68 Ga]Ga‐AJ206, with and without pre‐administration of a blocking dose (2 mg kg −1 of AJ206) (tumor denoted with dashed red line). E) [ 68 Ga]Ga‐AJ206 quantification in tumors by ex vivo biodistribution in mice treated with and without a blocking dose; data in figure C and E are shown as box and whisker plots showing all data points ( n = 4–5). Ordinary one‐way ANOVA using multiple comparison test in C and multiple unpaired t test in E. ns, P ≥ 0.05; *, P ≤ 0.05; ** P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: In Vivo, Positron Emission Tomography-Computed Tomography, Injection, Immunohistochemistry, Expressing, Ex Vivo, Blocking Assay, Whisker Assay, Comparison

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: Validation of [ 68 Ga]Ga‐AJ206 specificity for CD38 in disseminated MM disease models and primary plasma cell leukemia xenografts. A) IVIS‐bioluminescence image of luciferase expressing MM1S disseminated tumor model. B) In vivo uptake of [ 68 Ga]Ga‐AJ206 in lungs and bones in MM1S‐Luc bearing mice. C) Flow cytometry analysis of CD38 expression in lungs harvested from MM1s‐Luc cells injected mice. Lungs from PBS treated mice were used as controls (top panel). IHC staining of bones shows CD38 expression (bottom panel). D) Rendered in vivo and ex vivo [ 68 Ga]Ga‐AJ206‐PET images of lower limbs of MOLP8 injected animals. E) IHC images of MOLP8 bone marrow tumor and PBS‐treated animals confirmed CD38 expression. F) Quantification of PET signal (%ID/cc) in the bone marrow of PBS, MM1S‐Luc and MOLP8 injected animals. G) Tumor/muscle (T/M) ratios of PET measures in the bone marrow of PBS, MM1S and MOLP8 injected animals. H) Flow cytometry analysis of CD38 expression in cells extracted from the bone marrow of PBS, MM1S‐Luc and MOLP8 cell injected mice. Data in figure F and G are shown as box and whisker plots showing all data points ( n = 7 in PBS, n = 5 in MM1S and n = 8 in MOLP8). Ordinary one‐way ANOVA using multiple comparison test. **, P ≤ 0.01; *** P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: Biomarker Discovery, Clinical Proteomics, Luciferase, Expressing, In Vivo, Flow Cytometry, Injection, Immunohistochemistry, Ex Vivo, Whisker Assay, Comparison

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: Validation of [ 68 Ga]Ga‐AJ206 specificity for CD38 in primary plasma cell leukemia xenografts. A) Flow cytometry histograms of CD38 expression in primary cells. PDX‐1 is from the peripheral blood of a relapsed/refractory MM patient with secondary plasma cell leukemia and PDX‐2 is from a newly diagnosed MM patient bone marrow. Neither had exposure to anti‐CD38 therapies. B) Static whole‐body PET/CT images of PDX bearing mice at 60 min postinjection of [ 68 Ga]Ga‐AJ206. (tumor in dashed red lines) C) IHC analysis of CD38 expression in PDXs.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: Biomarker Discovery, Clinical Proteomics, Flow Cytometry, Expressing, Positron Emission Tomography-Computed Tomography

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: In vitro and in vivo detection of ATRA treatment induced changes in CD38 expression by [ 68 Ga]Ga‐AJ206 PET in MM cells and PDXs. A) Flow cytometry analysis of ATRA induced changes in surface expression of CD38 in MM cells. B) In vitro uptake of [ 68 Ga]Ga‐AJ206 (%IA) in MM cells treated with ATRA or vehicle control. Cells were incubated with [ 68 Ga]Ga‐AJ206 at 4 °C for 1 h. C) Static whole‐body PET/CT images of PDX bearing NSG mice before and after treatment with ATRA. Red circles indicate tumor. D) Quantification of PET signal in tumors pre‐ and post‐treatment with ATRA. E) Ratio of PET signal in tumors before ( U 0 ) and after ATRA ( U t ) treatment. F) Tumor/muscle ratio of PET measures before and after ATRA treatment G) CD38 IHC of PDXs of untreated and post‐ATRA treated mice; data in figures A and B are represented as mean ± SD ( n = 3 or 4) and significance was calculated by multiple unpaired t test; data in figure D–F are shown for individual mice and significance was calculated using paired t test . ns, P ≥ 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: In Vitro, In Vivo, Expressing, Flow Cytometry, Control, Incubation, Positron Emission Tomography-Computed Tomography

Journal: Advanced Science

Article Title: CD38‐Specific Gallium‐68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET

doi: 10.1002/advs.202308617

Figure Lengend Snippet: Parameters of affinity measurements of AJ206 by SPR.

Article Snippet: The ligands used were His‐Tagged human CD38 (R&D systems, catalog # 2404‐AC, 43 kDa, 0.5 mg mL −1 stock concentration) and mouse CD38 proteins (R&D systems, catalog # 4947‐AC, 40 kDa, 1.81 mg mL −1 stock concentration), which were immobilized onto the CM5 chip.

Techniques: Ligand Binding Assay

Journal: Autophagy

Article Title: LRRK2 is required for CD38-mediated NAADP-Ca 2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

doi: 10.1080/15548627.2021.1954779

Figure Lengend Snippet: Figure 1. Anti-CD38 mAb clone 90 drives CD38 internalization and TFEB nuclear translocation via NAADP-driven calcium signaling. (A) Immunoblot of endogenous TFEB after nuclear fractionation of WT B-lymphocytes treated with 10 µg/ml anti-CD38 antibody (clone 90) or an isotype control (Iso) for 1 h. (B) Quantification of nuclear TFEB normalized to histone H3 from (A) (n = 3). (C) Immunoblot of endogenous TFEB after nuclear fractionation of WT BMDMs treated with 5 µg/ml (+) or 10 µg/ml (++) anti-CD38 antibody (clone 90) for 1 h. (D) Quantification of nuclear TFEB normalized to histone H3 from (C) (n = 4). (E) Confocal imaging of WT BMDMs treated with 10 µg/ml anti-CD38 for 1 h and stained for TFEB (red) and CD38 (clone 90, green). Scale bar: 70 µm, 30 µm (zoom). (F) Confocal imaging of HEK293T cells transfected with HA-CD38 stimulated with anti-CD38 antibody (clone 90-Alexa Fluor 647, red) at either 4°C or 37°C for 30 min and subsequently stained for HA (green). Scale bar: 10 µm. (G) CD38 internalization after stimulation with anti-CD38 (clone 90) or an isotype control (10 µg/ml) (n = 2). (H) Confocal imaging of WT BMDMs following stimulation with anti-CD38 (clone 90, 10 µg/ml), an isotype control, or starvation. Quantification of nuclear:cytosolic mean fluorescence intensity (MFI) for each condition is shown (n ≥ 20 cells per condition). Scale bar: 20 µm. (I) Confocal imaging of WT BMDMs following stimulation with anti-CD38 (clone 90, 10 µg/ml) after preincubation with the indicated inhibitor. Quantification of nuclear:cytosolic mean fluorescence intensity (MFI) for each condition is shown (n ≥ 150 cells per condition). Scale bar: 30 µm. (J) Intracellular calcium response of WT B-lymphocytes stimulated with isotype control (10 µg/ml, blue) or anti-CD38 (clone 90 10 µg/ml, black) antibody with or without preincubation with Ned-19 (100 µM, green) or Gly-Phe-β-naphtylamide (GPN 200 µM, red). (K) Quantification of calcium responses (area under curve) to 5 min after stimulation with anti-CD38 (clone 90) and preincubation with Ned-19 (100 µM), 8-Br-ADPR (100 µM), 8-Br-cADPR (100 µM), GPN (200 µM), or EDTA (2 mM) (n = 3). Calcium response curve is shown to 5 min as fluorescent counts (iLm1). For western data, histone H3 and GAPDH are shown as fractionation controls. Western bands are quantified, normalized to the loading control, then presented relative to the control. (*p < 0.05; **p < 0.002, ***p < 0.0001, Student’s t-test or one-way ANOVA with post hoc Tukey’s HSD).

Article Snippet: The following primary antibodies were used for western blotting: phospho-LRRK2 (Ser935, UDD2 10 [12]; Abcam, ab133450; 1:1000), total LRRK2 (MJFF2; Abcam, ab133474; 1:1000), human TFEB (Bethyl Laboratories, A303-672A; 1:2000), human TFEB (Cell Signaling Technology, 4202; 1:1000), mouse TFEB (Bethyl Laboratories, A303-673A; 1:1000), phospho-serine (Q5; Qiagen, 37430; 1:800), phospho-serine (Q7; Qiagen, 37420; 1:800), LAMP1 (H4A3; Santa Cruz Biotechnology, sc20011; 1:1000), CTSD (C-20; Santa Cruz Biotechnology, sc377299; 1:1000), SQSTM1/p62 (P-15; Santa Cruz Biotechnology, sc-28359; 1:1000), BECN1 (K-15; Santa Cruz Biotechnology,sc-10087; 1:1000), MAP1LC3B (Cell Signaling Technology, 2775; 1:1000), CD38 (R&D Systems, AF4947; 1:2000), mouse-anti-FLAG (M2; Sigma-Aldrich, F1804; 1:1000), rabbit-anti-FLAG (Rockland, 600–401-383; 1:2000), mouse-anti-MYC (9E10; Santa Cruz Biotechnology, sc-40; 1:500), rabbit-anti-MYC (71D10; Cell Signaling Technology, 2278; 1:1000), GFP (Cell Signaling, 2555; 1:1000), phosphoPRKAA/AMPK (Thr172) (40H9; Cell Signaling Technology, 2535; 1:1000), PRKAA/AMPK (D5A2; Cell Signaling Technology, 5831; 1:2000), TPCN2 (Abcam, ab119915; 1:500), PPP3CB (Thermo Fisher Scientific, 55148-1-AP; 1:1000), MTOR (7 C10; Cell Signaling Technology, 2983; 1:1000), phospho-RPS6KB1/p70S6K (Thr389) (1A5; Cell Signaling Technology, 9206; 1:1000), total RPS6KB1/p70S6K (49D7; Cell Signaling Technology, 2708; 1:1000), phosphoRPS6/S6 ribosomal protein (Ser236/256) (Cell Signaling Technology, 2211S; 1:1000), phospho-GSK3B (Ser9) (Cell Signaling Technology, 9336; 1:1000), total GSK3B (1V001; Santa Cruz Biotechnology, sc-71186; 1:1000), phosphoMAPK/ERK (Thr202/Tyr204) (E10; Cell Signaling Technology, 9106; 1:1000), total MAPK/ERK (137 F5; Cell Signaling Technology, 4695; 1:1000), phospho-RAB8 (Thr72) (MJF-R20; Abcam, ab230260; 1:5000), total RAB8 (EPR14873; Abcam, ab188574; 1:2000), Histone H3 (Cell Signaling Technology, 4499; 1:2000), HRP-conjugated GAPDH (Proteintech Group, HRP-60004; 1:10,000), HRP conjugated ACTB/β-actin (Sigma-Aldrich, A3854; 1:10,000).

Techniques: Translocation Assay, Western Blot, Fractionation, Control, Imaging, Staining, Transfection, Fluorescence

Journal: Autophagy

Article Title: LRRK2 is required for CD38-mediated NAADP-Ca 2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

doi: 10.1080/15548627.2021.1954779

Figure Lengend Snippet: Figure 2. CD38 calcium signaling and nuclear localization of TFEB is dependent on LRRK2. Immunoblot of endogenous TFEB after nuclear fractionation of WT and lrrk2 KO (A) B-lymphocytes and (B) BMDMs treated with 5 µg/ml anti-CD38 antibody (clone 90) or an isotype control (Iso) for 1 h. (C) Quantification of western blots from A (upper, n = 4) and B (lower, n = 3). (D) Intracellular calcium response of purified WT (black) and lrrk2 KO (red) B-lymphocytes stimulated with anti-CD38 (clone 90, 5 and 10 µg/ml) antibody. (E) Quantification of calcium response (area under curve) from (D) to 5 min (n = 4). (F) Intracellular calcium response of purified WT (black) and LRRK2G2019S KI (blue) B-lymphocytes stimulated with anti-CD38 (clone 90, 5 and 10 µg/ml) antibody. (G) Quantification of calcium response from (F) to 5 min (n = 4). (H) Intracellular calcium response of purified WT (solid lines) or LRRK2G2019S KI (dashed lines) B-lymphocytes stimulated with anti-CD38 (clone 90 10 µg/ ml, black) antibody with or without Ned-19 (100 µM, blue) or Gly-Phe-β-naphthylamide (200 µM, green). (I) Quantification of calcium responses (area under curve) from (H) to 5 min (n = 4). (J) Purified wild type (WT, black) or lrrk2 KO (KO, red) B-lymphocytes stimulated with NAADP-AM (3 µM). (K) Quantification of calcium responses (area under curve) from (H) to 5 min (n = 6). Partial calcium response curve is shown (to 3 min) as fluorescent counts (iLm1). For western data, histone H3 and GAPDH are shown as fractionation and loading controls. Western bands are quantified, normalized to the loading control, and presented relative to the control lane. (# – not significant; *p < 0.05; **p < 0.002, ***p < 0.0001, Student’s t-test or one-way ANOVA with post hoc Tukey’s HSD or Fisher’s LSD).

Article Snippet: The following primary antibodies were used for western blotting: phospho-LRRK2 (Ser935, UDD2 10 [12]; Abcam, ab133450; 1:1000), total LRRK2 (MJFF2; Abcam, ab133474; 1:1000), human TFEB (Bethyl Laboratories, A303-672A; 1:2000), human TFEB (Cell Signaling Technology, 4202; 1:1000), mouse TFEB (Bethyl Laboratories, A303-673A; 1:1000), phospho-serine (Q5; Qiagen, 37430; 1:800), phospho-serine (Q7; Qiagen, 37420; 1:800), LAMP1 (H4A3; Santa Cruz Biotechnology, sc20011; 1:1000), CTSD (C-20; Santa Cruz Biotechnology, sc377299; 1:1000), SQSTM1/p62 (P-15; Santa Cruz Biotechnology, sc-28359; 1:1000), BECN1 (K-15; Santa Cruz Biotechnology,sc-10087; 1:1000), MAP1LC3B (Cell Signaling Technology, 2775; 1:1000), CD38 (R&D Systems, AF4947; 1:2000), mouse-anti-FLAG (M2; Sigma-Aldrich, F1804; 1:1000), rabbit-anti-FLAG (Rockland, 600–401-383; 1:2000), mouse-anti-MYC (9E10; Santa Cruz Biotechnology, sc-40; 1:500), rabbit-anti-MYC (71D10; Cell Signaling Technology, 2278; 1:1000), GFP (Cell Signaling, 2555; 1:1000), phosphoPRKAA/AMPK (Thr172) (40H9; Cell Signaling Technology, 2535; 1:1000), PRKAA/AMPK (D5A2; Cell Signaling Technology, 5831; 1:2000), TPCN2 (Abcam, ab119915; 1:500), PPP3CB (Thermo Fisher Scientific, 55148-1-AP; 1:1000), MTOR (7 C10; Cell Signaling Technology, 2983; 1:1000), phospho-RPS6KB1/p70S6K (Thr389) (1A5; Cell Signaling Technology, 9206; 1:1000), total RPS6KB1/p70S6K (49D7; Cell Signaling Technology, 2708; 1:1000), phosphoRPS6/S6 ribosomal protein (Ser236/256) (Cell Signaling Technology, 2211S; 1:1000), phospho-GSK3B (Ser9) (Cell Signaling Technology, 9336; 1:1000), total GSK3B (1V001; Santa Cruz Biotechnology, sc-71186; 1:1000), phosphoMAPK/ERK (Thr202/Tyr204) (E10; Cell Signaling Technology, 9106; 1:1000), total MAPK/ERK (137 F5; Cell Signaling Technology, 4695; 1:1000), phospho-RAB8 (Thr72) (MJF-R20; Abcam, ab230260; 1:5000), total RAB8 (EPR14873; Abcam, ab188574; 1:2000), Histone H3 (Cell Signaling Technology, 4499; 1:2000), HRP-conjugated GAPDH (Proteintech Group, HRP-60004; 1:10,000), HRP conjugated ACTB/β-actin (Sigma-Aldrich, A3854; 1:10,000).

Techniques: Western Blot, Fractionation, Control, Purification

Journal: Autophagy

Article Title: LRRK2 is required for CD38-mediated NAADP-Ca 2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

doi: 10.1080/15548627.2021.1954779

Figure Lengend Snippet: Figure 3. Clone 90 ligation targets CD38 to the endolysosomal system independently of LRRK2. (A) Confocal imaging analysis in WT and lrrk2 KO BMDMs to directly visualize colocalization between CD38 (tagged with clone 90-Alexa Fluor 647, red) in the absence of signaling (4°C) and after initiation of signaling for 30 min (37°C) with co-staining of LAMP1 (green). Colocalization channel is shown in gray pseudocolor. (B) Manders’ coefficient illustrating percent of CD38 colocalizing with LAMP1 (n ≥ 10). (C) Confocal imaging analysis in WT and lrrk2 KO BMDMs to directly visualize colocalization between CD38-Alexa Fluor 647 (red) 30 min after initiation of signaling with co-staining of RAB7 after cell fixation (green). (D) Manders’ coefficient illustrating percent of CD38 colocalizing with RAB7 (n ≥ 10). (# – not significant; *p < 0.05; **p < 0.002, ***p < 0.0001, Student’s t-test or one-way ANOVA with post hoc Tukey’s HSD).

Article Snippet: The following primary antibodies were used for western blotting: phospho-LRRK2 (Ser935, UDD2 10 [12]; Abcam, ab133450; 1:1000), total LRRK2 (MJFF2; Abcam, ab133474; 1:1000), human TFEB (Bethyl Laboratories, A303-672A; 1:2000), human TFEB (Cell Signaling Technology, 4202; 1:1000), mouse TFEB (Bethyl Laboratories, A303-673A; 1:1000), phospho-serine (Q5; Qiagen, 37430; 1:800), phospho-serine (Q7; Qiagen, 37420; 1:800), LAMP1 (H4A3; Santa Cruz Biotechnology, sc20011; 1:1000), CTSD (C-20; Santa Cruz Biotechnology, sc377299; 1:1000), SQSTM1/p62 (P-15; Santa Cruz Biotechnology, sc-28359; 1:1000), BECN1 (K-15; Santa Cruz Biotechnology,sc-10087; 1:1000), MAP1LC3B (Cell Signaling Technology, 2775; 1:1000), CD38 (R&D Systems, AF4947; 1:2000), mouse-anti-FLAG (M2; Sigma-Aldrich, F1804; 1:1000), rabbit-anti-FLAG (Rockland, 600–401-383; 1:2000), mouse-anti-MYC (9E10; Santa Cruz Biotechnology, sc-40; 1:500), rabbit-anti-MYC (71D10; Cell Signaling Technology, 2278; 1:1000), GFP (Cell Signaling, 2555; 1:1000), phosphoPRKAA/AMPK (Thr172) (40H9; Cell Signaling Technology, 2535; 1:1000), PRKAA/AMPK (D5A2; Cell Signaling Technology, 5831; 1:2000), TPCN2 (Abcam, ab119915; 1:500), PPP3CB (Thermo Fisher Scientific, 55148-1-AP; 1:1000), MTOR (7 C10; Cell Signaling Technology, 2983; 1:1000), phospho-RPS6KB1/p70S6K (Thr389) (1A5; Cell Signaling Technology, 9206; 1:1000), total RPS6KB1/p70S6K (49D7; Cell Signaling Technology, 2708; 1:1000), phosphoRPS6/S6 ribosomal protein (Ser236/256) (Cell Signaling Technology, 2211S; 1:1000), phospho-GSK3B (Ser9) (Cell Signaling Technology, 9336; 1:1000), total GSK3B (1V001; Santa Cruz Biotechnology, sc-71186; 1:1000), phosphoMAPK/ERK (Thr202/Tyr204) (E10; Cell Signaling Technology, 9106; 1:1000), total MAPK/ERK (137 F5; Cell Signaling Technology, 4695; 1:1000), phospho-RAB8 (Thr72) (MJF-R20; Abcam, ab230260; 1:5000), total RAB8 (EPR14873; Abcam, ab188574; 1:2000), Histone H3 (Cell Signaling Technology, 4499; 1:2000), HRP-conjugated GAPDH (Proteintech Group, HRP-60004; 1:10,000), HRP conjugated ACTB/β-actin (Sigma-Aldrich, A3854; 1:10,000).

Techniques: Ligation, Imaging, Staining

Journal: Autophagy

Article Title: LRRK2 is required for CD38-mediated NAADP-Ca 2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

doi: 10.1080/15548627.2021.1954779

Figure Lengend Snippet: Figure 4. CD38 and LRRK2 interact after CD38 activation. (A) Co-immunoprecipitation of endogenous CD38 and LRRK2 in purified wild type B-lymphocytes following pulldown of anti-CD38 (clone 90, 10 µg/ml)-CD38 complexes using protein G affinity isolation after stimulation for the indicated time. (B) Schematic of LRRK2 constructs used in mapping the CD38-LRRK2 interaction (LRR: Leucine-rich repeat, ROC: Ras of complex, COR: C-terminal of ROC). (C and D) Domain mapping of the interaction between CD38 and LRRK2 using the indicated constructs. (E) Fluorescence resonance electron transfer fluorescence lifetime imaging (FRET-FLIM) of HEK293T cells transfected with MYC-LRRK2 with or without HA-CD38 imaged unstimulated or after anti-CD38 (clone 90, 10 µg/ml) stimulation for 30 min. Top panel shows fluorescence intensity of MYC-LRRK2-594, while bottom panels depict fluorescence lifetime with MYC-LRRK2-594 functioning as the donor and clone 90-Alexa Fluor 647 as the acceptor. White arrows indicate areas of CD38-LRRK2 interaction. Scale bar: 10 μm, 3.75 μm (zoom). (F) Quantification of the average fluorescence lifetime of MYC-LRRK2-594 (n = 15). (G) Calculated FRET efficiency in the unstimulated or clone-90 stimulated state. (H) Immunoblotting of the indicated proteins after stimulation with anti-CD38 (clone 90, 10 µg/ml) for the indicated time. ACTB/β-Actin is shown as a loading control. (# – not significant, *p < 0.05; **p < 0.002, ***p < 0.0001, Student’s t-test or one-way ANOVA with post hoc Tukey’s HSD).

Article Snippet: The following primary antibodies were used for western blotting: phospho-LRRK2 (Ser935, UDD2 10 [12]; Abcam, ab133450; 1:1000), total LRRK2 (MJFF2; Abcam, ab133474; 1:1000), human TFEB (Bethyl Laboratories, A303-672A; 1:2000), human TFEB (Cell Signaling Technology, 4202; 1:1000), mouse TFEB (Bethyl Laboratories, A303-673A; 1:1000), phospho-serine (Q5; Qiagen, 37430; 1:800), phospho-serine (Q7; Qiagen, 37420; 1:800), LAMP1 (H4A3; Santa Cruz Biotechnology, sc20011; 1:1000), CTSD (C-20; Santa Cruz Biotechnology, sc377299; 1:1000), SQSTM1/p62 (P-15; Santa Cruz Biotechnology, sc-28359; 1:1000), BECN1 (K-15; Santa Cruz Biotechnology,sc-10087; 1:1000), MAP1LC3B (Cell Signaling Technology, 2775; 1:1000), CD38 (R&D Systems, AF4947; 1:2000), mouse-anti-FLAG (M2; Sigma-Aldrich, F1804; 1:1000), rabbit-anti-FLAG (Rockland, 600–401-383; 1:2000), mouse-anti-MYC (9E10; Santa Cruz Biotechnology, sc-40; 1:500), rabbit-anti-MYC (71D10; Cell Signaling Technology, 2278; 1:1000), GFP (Cell Signaling, 2555; 1:1000), phosphoPRKAA/AMPK (Thr172) (40H9; Cell Signaling Technology, 2535; 1:1000), PRKAA/AMPK (D5A2; Cell Signaling Technology, 5831; 1:2000), TPCN2 (Abcam, ab119915; 1:500), PPP3CB (Thermo Fisher Scientific, 55148-1-AP; 1:1000), MTOR (7 C10; Cell Signaling Technology, 2983; 1:1000), phospho-RPS6KB1/p70S6K (Thr389) (1A5; Cell Signaling Technology, 9206; 1:1000), total RPS6KB1/p70S6K (49D7; Cell Signaling Technology, 2708; 1:1000), phosphoRPS6/S6 ribosomal protein (Ser236/256) (Cell Signaling Technology, 2211S; 1:1000), phospho-GSK3B (Ser9) (Cell Signaling Technology, 9336; 1:1000), total GSK3B (1V001; Santa Cruz Biotechnology, sc-71186; 1:1000), phosphoMAPK/ERK (Thr202/Tyr204) (E10; Cell Signaling Technology, 9106; 1:1000), total MAPK/ERK (137 F5; Cell Signaling Technology, 4695; 1:1000), phospho-RAB8 (Thr72) (MJF-R20; Abcam, ab230260; 1:5000), total RAB8 (EPR14873; Abcam, ab188574; 1:2000), Histone H3 (Cell Signaling Technology, 4499; 1:2000), HRP-conjugated GAPDH (Proteintech Group, HRP-60004; 1:10,000), HRP conjugated ACTB/β-actin (Sigma-Aldrich, A3854; 1:10,000).

Techniques: Activation Assay, Immunoprecipitation, Purification, Isolation, Construct, Fluorescence, Imaging, Transfection, Western Blot, Control

Journal: Autophagy

Article Title: LRRK2 is required for CD38-mediated NAADP-Ca 2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

doi: 10.1080/15548627.2021.1954779

Figure Lengend Snippet: Figure 8. Schematic illustrating the CD38-LRRK2-TFEB signaling pathway in B-lymphocytes and macrophages. The CD38-LRRK2 complex exists on the plasma membrane and activation of CD38 results in the internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. CD38 promotes generation of an NAADP-dependent lysosomal calcium signal, which is dependent on LRRK2 kinase activity. This results in the activation of PPP3/calcineurin, which dephosphorylates TFEB at Ser211 and allows its nuclear translocation. The pathogenic kinase overactive LRRK2G2019S mutant aberrantly activates TFEB via NAADP- TPCN2-dependent calcium signaling and stabilizes TFEB by promoting its C-terminal phosphorylation. Solid arrows indicate known relationships, while dashed arrows indicated hypothesized connections.

Article Snippet: The following primary antibodies were used for western blotting: phospho-LRRK2 (Ser935, UDD2 10 [12]; Abcam, ab133450; 1:1000), total LRRK2 (MJFF2; Abcam, ab133474; 1:1000), human TFEB (Bethyl Laboratories, A303-672A; 1:2000), human TFEB (Cell Signaling Technology, 4202; 1:1000), mouse TFEB (Bethyl Laboratories, A303-673A; 1:1000), phospho-serine (Q5; Qiagen, 37430; 1:800), phospho-serine (Q7; Qiagen, 37420; 1:800), LAMP1 (H4A3; Santa Cruz Biotechnology, sc20011; 1:1000), CTSD (C-20; Santa Cruz Biotechnology, sc377299; 1:1000), SQSTM1/p62 (P-15; Santa Cruz Biotechnology, sc-28359; 1:1000), BECN1 (K-15; Santa Cruz Biotechnology,sc-10087; 1:1000), MAP1LC3B (Cell Signaling Technology, 2775; 1:1000), CD38 (R&D Systems, AF4947; 1:2000), mouse-anti-FLAG (M2; Sigma-Aldrich, F1804; 1:1000), rabbit-anti-FLAG (Rockland, 600–401-383; 1:2000), mouse-anti-MYC (9E10; Santa Cruz Biotechnology, sc-40; 1:500), rabbit-anti-MYC (71D10; Cell Signaling Technology, 2278; 1:1000), GFP (Cell Signaling, 2555; 1:1000), phosphoPRKAA/AMPK (Thr172) (40H9; Cell Signaling Technology, 2535; 1:1000), PRKAA/AMPK (D5A2; Cell Signaling Technology, 5831; 1:2000), TPCN2 (Abcam, ab119915; 1:500), PPP3CB (Thermo Fisher Scientific, 55148-1-AP; 1:1000), MTOR (7 C10; Cell Signaling Technology, 2983; 1:1000), phospho-RPS6KB1/p70S6K (Thr389) (1A5; Cell Signaling Technology, 9206; 1:1000), total RPS6KB1/p70S6K (49D7; Cell Signaling Technology, 2708; 1:1000), phosphoRPS6/S6 ribosomal protein (Ser236/256) (Cell Signaling Technology, 2211S; 1:1000), phospho-GSK3B (Ser9) (Cell Signaling Technology, 9336; 1:1000), total GSK3B (1V001; Santa Cruz Biotechnology, sc-71186; 1:1000), phosphoMAPK/ERK (Thr202/Tyr204) (E10; Cell Signaling Technology, 9106; 1:1000), total MAPK/ERK (137 F5; Cell Signaling Technology, 4695; 1:1000), phospho-RAB8 (Thr72) (MJF-R20; Abcam, ab230260; 1:5000), total RAB8 (EPR14873; Abcam, ab188574; 1:2000), Histone H3 (Cell Signaling Technology, 4499; 1:2000), HRP-conjugated GAPDH (Proteintech Group, HRP-60004; 1:10,000), HRP conjugated ACTB/β-actin (Sigma-Aldrich, A3854; 1:10,000).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Activity Assay, Translocation Assay, Mutagenesis, Phospho-proteomics

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Reduced ALDH1A1 expression in multiple myeloma cells increases resistance to daratumumab via downregulation of retinoic acid

doi: 10.1007/s00018-025-05891-7

Figure Lengend Snippet: ALDH1A1 increased after conventional chemotherapy regimen Vrd or Krd. A and B. GSE276561 and GSE47552 datasets revealed ALDH1A1 in MM was lower than healthy control; These GEO data were analyzed by on line tool GEO2R using Benjamini & Hochberg (False discovery rate) method; Each y axis label in the graph represents the expression measurement extracted from the TPM normalized expression counts (for RNA-seq), or the Value column of the original submitter-supplied Sample record (for microarrays). All the y axis label represents normalized mRNA expression level. C. mRNA levels of ALDH1A1 in NDMM samples are significantly lower than healthy control, but remarkably higher in RRMM samples than NDMM patients which are collected in our hospital; Relative ALDH1A1 levels is relative to GAPDH. D. mRNA levels of CD38 in NDMM patients are significantly higher than healthy control which are collected in our hospital; Relative CD38 levels is relative to GAPDH. E. ALDH1A1 mRNA increased after Vrd or Krd treatment; Relative ALDH1A1 levels is relative to GAPDH. F . western blot confirmed ALDH1A1 protein increased after Vrd or Krd treatment. **, p < 0.01, ***, p < 0.001. C-E, Data was analyzed by Mann-Whitnay test HC: healthy control; Vrd: Bortezomib + Lenalidomide + Dexamethasone; KRd: Carfilzomib + Lenalidomide + Dexamethasone; MM: multiple myeloma; NDMM: newly diagnosed multiple myeloma; SMM: smoldering multiple myeloma; MGUS: monoclonal gammopathy of undetermined significance

Article Snippet: The positive CD38 cells were labeled by APC anti-human CD38 antibody (E-AB-F1058E, Elabscience) or APC anti-mouse CD38 antibody (E-AB-F1193E, Elabscience) and analyzed by FACSCantoTM II flow cytometer.

Techniques: Control, Expressing, RNA Sequencing, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Reduced ALDH1A1 expression in multiple myeloma cells increases resistance to daratumumab via downregulation of retinoic acid

doi: 10.1007/s00018-025-05891-7

Figure Lengend Snippet: ALDH1A1 down-regulated in the nrRRMM patients received Dara-Rd treatment. A and B. rRRMM patients had higher ALDH1A expression compared with nrRRMM after Dara-Rd treatment analyzed by RT-qPCR and western blot; C and D . the CD38 expression (mRNA and protein) decreased in Dara-treated patients in nrRRMM patients compared with rRRMM patients; E . the mRNA expression of ALDH1A1 after Dara-based treatment was positively correlated with the CD38 mRNA expression. **, p < 0.01 A-D, data was analyzed by student t test. E, data was analyzed by simple linear regression nrRRMM: non-responder relapsed/refractory multiple myeloma; rRRMM: responder relapsed/refractory multiple myeloma; MFI: median fluorescence intensity; Dara-Rd: daratumumab + lenalidomide + dexamethasone; Dara: daratumumab

Article Snippet: The positive CD38 cells were labeled by APC anti-human CD38 antibody (E-AB-F1058E, Elabscience) or APC anti-mouse CD38 antibody (E-AB-F1193E, Elabscience) and analyzed by FACSCantoTM II flow cytometer.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Reduced ALDH1A1 expression in multiple myeloma cells increases resistance to daratumumab via downregulation of retinoic acid

doi: 10.1007/s00018-025-05891-7

Figure Lengend Snippet: ALDH1A1 directly regulates CD38 expression in H929 cells. A . the KD efficiency of different sh-ALDH1A1 sequences; B . Knock down ALDH1A1 decreased CD38 genes expression; C. Re-introducing ALDH1A1-OE plasmid restored downregulation of ALDH1A1 mRNA levels in ALDH1A1-KD cells; D . Re-introducing ALDH1A1 into ALDH1A1-KD cells significantly upregulate the expression of CD38; E . cell-surface expression of CD38 decreased in ALDH1A1-KD cells but restored by re-introducing ALDH1A1-OE plasmid determined by flow cytometry analysis. **, p < 0.01, ***, p < 0.001 Student t test was performed in all datasets AlDH1A1-KD: ALDH1A1 knockdown; Ctrl: control; shALDH1A1: short hairpin RNA for ALDH1A1; KD-OE: over expression of ALDH1A1 in KD cells

Article Snippet: The positive CD38 cells were labeled by APC anti-human CD38 antibody (E-AB-F1058E, Elabscience) or APC anti-mouse CD38 antibody (E-AB-F1193E, Elabscience) and analyzed by FACSCantoTM II flow cytometer.

Techniques: Expressing, Knockdown, Plasmid Preparation, Flow Cytometry, Control, shRNA, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Reduced ALDH1A1 expression in multiple myeloma cells increases resistance to daratumumab via downregulation of retinoic acid

doi: 10.1007/s00018-025-05891-7

Figure Lengend Snippet: ALDH1A1 increased CD38 through activation of RA/RAR via RA production. A . Volcano plots created by differential mRNA (fold change (FC) > 2 or < 0.5, and p < 0.05); B. KEGG analysis in ALDH1A1 KD cells of H929 and 8226 cells revealed that retinoic acid receptor, and retinoid X receptor agonists/antagonists pathways were enriched in both cell line; C. Knock down ALDH1A1caused downregulation of RAR proteins while re-introduced ALDH1A1 into ALDH1A1-KD cells increased their expressions; D . RA salvage restore the ADCC of Dara against MM cells; E . the RAR protein level was significantly increased determined by western blot after RA supplement; (F) mRNA levels of CD38 determined by RT-qPCR; (G) protein levels of CD38 determined by western blot. **, p < 0.01, ***, p < 0.001 C-G, student t test was adopted KD: ALDH1A1 knockdown; Ctrl: control; KD-OE: over expression of ALDH1A1 in KD cells; RA: retinoic acid; RAR: retinoic acid receptor; ADCC: antibody-dependent cellular cytotoxicity

Article Snippet: The positive CD38 cells were labeled by APC anti-human CD38 antibody (E-AB-F1058E, Elabscience) or APC anti-mouse CD38 antibody (E-AB-F1193E, Elabscience) and analyzed by FACSCantoTM II flow cytometer.

Techniques: Activation Assay, Knockdown, Western Blot, Quantitative RT-PCR, Control, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Reduced ALDH1A1 expression in multiple myeloma cells increases resistance to daratumumab via downregulation of retinoic acid

doi: 10.1007/s00018-025-05891-7

Figure Lengend Snippet: ALDH1A1 inhibitor reduces the sensitivity of Dara in vivo through inhibiting the RA/RAR pathway. A and B . compared with vehicle control, Dara cause significantly reduction of xenograft tumors weight and tumor growth; C . inhibitor of ALDH1A 673 A remarkably reduce the protein level of CD38, ALDH1A1 and RAR, and RA complement significantly restore the protein level of CD38 and RAR. *, p < 0.05, **, p < 0.01, ***, p < 0.001 A-B, analysis of variance (ANOVA) was used for multi-group comparisons followed by Tukey’s post hoc test ( N = 6); C, analysis of variance (ANOVA) was used for multi-group comparisons followed by Tukey’s post hoc test ( N = 3) RA: retinoic acid; RAR: retinoic acid receptor

Article Snippet: The positive CD38 cells were labeled by APC anti-human CD38 antibody (E-AB-F1058E, Elabscience) or APC anti-mouse CD38 antibody (E-AB-F1193E, Elabscience) and analyzed by FACSCantoTM II flow cytometer.

Techniques: In Vivo, Control

Journal: Cancer Cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD38 171Yb clone 90 , Fluidigm , 3171007B.

Techniques: Blocking Assay, Virus, Recombinant, Plasmid Preparation, Saline, Lysis, Staining, Reverse Transcription, In Vivo, Electroporation, Mass Cytometry, Conjugation Assay, Illumina Sequencing, Library Quantification, In Vitro, Quantitative RT-PCR, Software, Real-time Polymerase Chain Reaction, Cytometry, Microscopy, Imaging, Spectrophotometry