Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 1. TrkB receptors interact and colocalize with CPE. A, B, Interaction between TrkB receptors and CPE was assessed in HEK293 overexpressing cDNAs encoding Flag-TrkB, Myc-CPE, and empty vector. Cell lysates were immunoprecipitated (IP) with anti-Flag antibodies (A) or anti-Myc antibodies (B) and immunoblotting (IB) analysis was performed to detect immunoprecipi- tated proteins. C, Endogenous association of CPE and TrkB receptors. Mice hippocampal tissues lysates were subjected to immunoprecipitation with polyclonal rabbit anti-TrkB antibody or con- trol IgG, followed by immunoblotting with mouse anti-TrkB and mouse anti-CPE antibodies, respectively. A–C, Each co-IP experiment was repeated three times, and representative data are shown. D, Representative immunofluorescence images of the subcellular colocalization of Flag-TrkB and Myc-CPE in hippocampal neurons by confocal microscopy. Lower panels are enlarged images of the framed regions with the white arrows indicating the colocalization of Flag-TrkB and Myc-CPE as shown in yellow. Scale bar, 10mm. E, Hippocampal neuron cotransfected at DIV 7 with TrkB-GFP and CPE-RFP. Time-lapse images of transport vesicles were acquired at one frame every 20 s for 700 s. F, Corresponding kymographs showing overlapping trajectories of vesicles labeled for TrkB-GFP and CPE-RFP. G, Time-lapse sequence showing a moving vesicle that contains both TrkB-GFP and CPE-RFP, indicated with arrows. Scale bar, 5 mm.

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Confocal Microscopy, Labeling, Sequencing

Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 2. CPE mediates activity-dependent TrkB membrane surface insertion in hippocampal neurons. A, PC12 cell were transiently transfected with Ctr siRNA or CPE siRNA. After 2 d, cells lysates were immunoblotted with CPE antibody to detect the CPE-knockdown efficiency. Endogenous b -tubulin was used as an expression control. B, Representative immunofluorescence images of TrkB surface levels in neurons (9 DIV) expressing the indicated constructs with or without Gly treatment (200 mM, 10min). Surface TrkB levels were determined using ratiometric flu- orescence assay. The siRNA constructs all expressed fused RFP, and we only captured images of RFP-positive neuron (pseudo blue color). The total TrkB receptors were fused with GFP (green color). The surface TrkB stained with M2 antibody followed by Cy5-conjugated goat anti-mouse antibody (pseudo red color). Anti-FLAG antibody labeled surface TrkB receptors, and the GFP flu- orescence represented total receptor levels. Scale bar, 10mm. C, Surface levels of TrkB receptor were analyzed as in B. Relative surface levels were normalized to that of TrkB-GFP (two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 8.627, Ctr-Ctr siRNA vs Gly-Ctr siRNA ppp = 0.0042; n = 4). D, Surface levels of TrkB and T1 receptors were evaluated by surface biotinylation assay in hippocampal neurons transfected with the indicated siRNA. Surface labeled TrKB were detected by streptavidin pull-down followed by anti-TrkB immunoblotting. E, Quantification of the surface levels of TrkB receptors by the ratio of surface to total TrkB intensity normalized to the control group (two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 7.75, Ctr-FAM-Con VS Gly-FAM-CPE siRNA ppp = 0.0019; n = 4). F, Quantification of the total TrkB receptors normalized to the control group. Error bars indicate 6 SEM. G, Quantification of the surface levels of T1 receptors by the ratio of surface to total T1 intensity normalized to the control group. H, Representative epifluorescence images from the TrkB inter- nalization assay. The Control, Ctr siRNA, and CPE siRNA constructs all express nonfused GFP, and we only captured images of GFP-positive neurons (GFP image not shown). The total pool of Flag-TrkB initially present at the cell surface was labeled with 594-M2 (Internal 1 surface group, green, pseudo color). After BDNF treatment at 37°C for 15min, neurons were fixed by 4% PFA under nonpermeabilizing conditions, and the receptors remaining at the cell surface were labeled with Cy5-conjugated secondary antibody (surface group, red, pseudo color). Scale bar, 10mm. I, Quantitation of internalized TrkB levels in H was performed. All of the data are presented as mean 6 SEM determined from analysis of more than three independent experiments (n 25 cells for each condition per experiment).

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Activity Assay, Membrane, Transfection, Knockdown, Expressing, Control, Immunofluorescence, Construct, Staining, Labeling, Surface Biotinylation Assay, Western Blot, Quantitation Assay

Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 3. CPE regulates cLTP-enhanced TrkB surface trafficking and translocation into spine. A, Representative TIRF images are shown for hippocampal neurons coexpression of CPE-RFP and TrkB-GFP, in which yellow color points the doubled positive of CPE and TrkB. Scale bar, 2mm. B, Colocalization of CPE and TrkB was quantified (unpaired t test, t = 5.784, df = 4, ppp = 0.0203; n = 4). C, Representative line plot analysis of TrkB receptor (594 signal, red color) across a spine and the adjacent dendritic shaft of siRNA-GFP transfected neurons, as indicated. Values for TrkB receptors were taken from the fluorescence intensity peaks after background subtraction (yellow line). Scale bar, 2mm. D, Representative immunofluorescence images of total TrkB re- ceptor across a spine and the adjacent dendritic shaft of siRNA-GFP transfected neurons on cLTP stimuli. E, Quantitative analysis of the ratio of TrkB fluorescence in spines and dendrites after normalization (15 dendrites/cell, 15–18 mm2/dendrite). Two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 104.4, ppppp , 0.0001; n = 4. All the data are presented as mean 6 SEM (n 25 cells for each condition per experiment).

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Translocation Assay, Transfection, Fluorescence, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 4. Identification of the key binding domain in TrkB interacting with CPE that mediated activity-dependent surface TrkB recruitment. A, Coimmunoprecipitation was performed in HEK 293 cell expressing Myc-CPEC25-RFP and Flag-TrkB mutants. Cell lysates were immunoprecipitated using anti-Flag antibody, followed by immunoblotting with Myc antibodies, and Myc-RFP is a control protein to confirm RFP couldn’t combine with TrkB. B, Co-IP of Myc-CPEC25 and FLAG-TrkB deletion mutants as indicated in HEK 293 cells. C, Summary of TrkB mutants used in B and a summary of the CPEC25 interactions with the mutants of TrkB. FL, full-length domain; EX, Extracellular domain. D, Co-IP of Myc-CPEC25 and Flag-TrkB deletion mutants as indicated in HEK293 cells. E, Summary of TrkB mutants used in D and a summary of the CPEC25 interactions with the mutants of TrkB. A–E, Each co-IP experiment was repeated at least three times, and representa- tive data are shown. F, Representative immunofluorescence images of TrkB mutants or T1 chimeras surface levels in transfected hippocampal neurons before and after glycine treatment. Scale bar, 10mm. G, Quantification of surface levels of TrkB mutants by the ratio of red to green fluorescence intensity in F (two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 7.75, Ctr-TrkB FL versus Gly-TrkB FL ppp = 0.0063; Ctr-TrkBDBOX1 versus Gly-TrkBDBOX1 pp = 0.0174; Ctr-TrkBDBOX3 versus Gly-TrkBDBOX3 pp = 0.0492; n = 4). H, Quantification of sur- face levels of T1 chimeras by the ratio of red to green fluorescence intensity in F (two-way ANOVA with Bonferroni’s multiple comparisons test, F(3,24) = 3.845, pppp = 0.0007; n = 4). All the data are presented as mean 6 SEM (n 25 cells for each condition per experiment).

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Binding Assay, Activity Assay, Expressing, Immunoprecipitation, Western Blot, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Transfection, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 5. Identification of the key binding domain in CPE interacting with TrkB that mediated activity-dependent surface TrkB recruitment. A, Co-IP was performed in lysates of HEK 293 cells expressing Flag-TrkBDEX and Myc-CPEC25 truncated mutants. B, Summary of CPE-truncated mutants used in A and a summary of the TrkBDEX interactions with the mutants of CPE. C, Ratiometric fluorescence assay was performed to examine the influence of CPEC25D10-RFP on cLTP-induced surface recruitment of TrkB receptors. Representative epifluorescence images were shown. Scale bar, 10mm. The Control and CPEC25D10-RFP constructs all express fused RFP, and we only captured images of RFP-positive neurons (pseudo blue color). The total TrkB receptors were fused with GFP (green color). The surface TrkB stained with M2 antibody followed by Cy5-conjugated goat anti-mouse antibody (pseudo red color). D, Quantification of TrkB surface levels by the ratio of red to green fluorescence intensity in C (two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 4.193, pp = 0.0225; n = 4). E, TAT-CPE452-466 inhibits the interac- tion between CPEC25 and TrkBDEX. HEK293 cells were transfected with Myc-CPEC25 and FLAG-TrkBDEX. After 2 d cells were treated with TAT-Con or TAT-CPE452-466 (5 mM) for 30min before lysed. The interaction between CPEC25 and TrkBDEX was analyzed by IP using anti-Flag antibodies and followed by immunoblotting with Myc antibodies. F, Ratiometric fluorescence assay was performed to examine the influence of TAT-CPE452-466 peptides on glycine-induced surface recruitment of TrkB receptor. Representative images from the ratiometric fluorescence assay are shown. Scale bar, 10mm. G, Quantification of TrkB surface levels by the ratio of red to green fluorescence intensity in F (two-way ANOVA with Bonferroni’s multiple comparisons test, F(1,12) = 3.266, pp = 0.0481; n = 4). All the data are presented as mean 6 SEM (n 25 cells for each condition per experiment).

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Binding Assay, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Fluorescence, Control, Construct, Staining, Transfection, Western Blot

Journal: The Journal of Neuroscience

Article Title: Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory

doi: 10.1523/jneurosci.0236-21.2021

Figure Lengend Snippet: Figure 9. TAT-CPE452-466 blocks the interaction of TrkB and CPE in the DH and inhibits TrkB phosphorylation induced by CFC training. A, TAT-CPE452-466 blocks the interaction between TrkB and CPE. Co-IP was performed 1 h after CFC training with microinjection of TAT-Con or TAT-CPE452-466 into DH 30min before CFC training with the control of home cage (HC) group. One-way ANOVA with Tukey’s multiple comparisons, F(2,9) = 15.77, pppp = 0.0009, pp = 0.0375; n = 4/group. B, TAT-CPE452-466 inhibits the CFC-induced TrkB phosphorylation in DH. Phosphorylation and total protein levels of TrkB were assessed by Western blot 1 h after CFC training with microinjection of TAT-Con or TAT-CPE452-466 into DH 30 min before CFC training. One-way ANOVA with Tukey’s multiple comparisons, F(2,9) = 11.86, ppp = 0.0024, pp = 0.0445; n 4/group. C, Coronal sections of DG were stained with DAPI and the antibody to a pan-neuronal marker, NeuN, from the group 1 h after CFC training with microinjection of TAT-Con or TAT-CPE452-466 into DH 30 min before CFC training. Scale bar, 20mm. D, Spine density in basal dendrites of DG pyrami- dal neurons in the group 1 h after CFC training with microinjection of TAT-Con or TAT-CPE452-466 into DH 30min before CFC training. Scale bar, 10mm. E, Examples and Sholl analyses of Golgi- stained dentate gyrus neurons from the group 1 h after CFC training with microinjection of TAT-Con or TAT-CPE452-466 into DH 30min before CFC training.

Article Snippet: The following antibodies were used: rabbit anti-Flag from Thermo Fisher Scientific; rabbit anti c-Myc antibodies from Bethyl Laboratories; rabbit anti-TrkB antibody from Millipore; goat anti-TrkB antibodies from R&D Systems; mouse anti-CPE antibodies from BD Transduction Laboratories; rabbit IgG Sepharose, mouse anti-Flag (M2) monoclonal antibody and mouse anti-tubulin antibodies from Sigma-Aldrich; rabbit anti-p44/42 MAP kinase, mouse anti-phospho-p44/42 (Erk1/2; Thr202/ Tyr204), mouse anti-phospho-Akt (Ser473) and rabbit anti-pTrkB antibodies from Cell Signaling Technology; mouse anti-Akt1(B-1) antibody from Santa Cruz Biotechnology; chicken anti-TrkB In pAb from Promega; and horseradish peroxidas-conjugated goat anti-mouse or rabbit IgG and horseradish peroxidase-conjugated rabbit anti-goat IgG purchased from Calbiochem.

Techniques: Phospho-proteomics, Co-Immunoprecipitation Assay, Microinjection, Control, Western Blot, Staining, Marker

Journal: iScience

Article Title: Local keratinocyte-nociceptor interactions enhance obesity-mediated painful small fiber neuropathy via NGF-TrkA-PI3K signaling axis

doi: 10.1016/j.isci.2025.112047

Figure Lengend Snippet:

Article Snippet: Staining was performed using Mouse anti-Tuj1 (Tubulin b3, TUBB3) antibody (BioLegend, 801202, 1:200), Goat anti-TrkA antibody (R&D Systems, AF1056-SP, 1:100), Goat anti-TrkB antibody (R&D Systems, AF1494-SP, 1:100), and Goat anti-TrkC antibody (R&D Systems, AF1404-SP, 1:100) to detect axons, Chick anti-GFP antibody (abcam, ab13970, 1:300) to detect GCaMP3 protein in sensory axons, Rabbit anti-MBP (Myelin Basic Protein) antibody (abcam, ab40390, 1:200) to detect myelin membrane in peripheral nerves, Rabbit anti-K10 (Keratin 10) antibody (BioLegend, 905403, 1:400) and Guinea pig anti-K14 (Keratin 14) antibody (PROGEN, GP-CK14, 1:100) to detect keratinocytes, Armenian hamster anti-PECAM-1 antibody (Millipore Sigma, MAB1398Z, 1:300) and Rat anti-PLVAP antibody (BD Biosciences, 553849, 1:100) to detect endothelial cells, and Rat anti-CD45 antibody (eBioscience, 140451-85, 1:100) to detect hematopoietic cells.

Techniques: Recombinant, Quantitative RT-PCR, Software

Journal: Nature Communications

Article Title: LILRB2/PirB mediates macrophage recruitment in fibrogenesis of nonalcoholic steatohepatitis

doi: 10.1038/s41467-023-40183-3

Figure Lengend Snippet: a Nuclear translocation of P65 in MDMs. Scale bar, 50 µm. b Western blot analysis of the phosphorylation of signaling molecules in MDMs. c Assessment of the tyrosine phosphorylation of the PirB protein and interactions between PirB and SHP-1/2 by Co-IP assays. d Western blot analysis of the phosphorylation of signaling molecules in MDMs pretreated with indicated antibodies. e Effect of anti-PirB antibody on cytokine expression ( n = 12 cells examined over four independent experiments). f Effect of neutralizing antibodies against cytokines and PirB on lipogenesis and apoptosis of hepatocytes cocultured with MDMs in the presence of rA8 and PA ( n = 4 independent experiments). g Cytokine expression of MDMs from WT or PirB −/− mice after rA8 or scrambled protein treatment ( n = 6 cells examined over three independent experiments). h Effect of anti-PirB antibody and anti-integrin-α5β1 antibody on ANGPTL8-induced MDM migration ( n = 6 cells examined over three independent experiments). Scale bar, 200 µm. i PirB −/− MDMs were resistant to ANGPTL8-induced migration ( n = 6 independent experiments). j Migration of MDMs pretreated with phosphorylation inhibitors before rA8 or scrambled protein stimulation ( n = 6 cells examined over three independent experiments). U0126, p-ERK1/2 inhibitor; SB203580, p-P38 inhibitor; MK-2206, p-AKT inhibitor; BAY11-7082, p-P65 inhibitor. k Experimental scheme to develop BM chimeras. l Flow cytometry analysis and quantification of KCs (CLEC2 hi F4/80 hi CD11b lo ) and MDMs (CLEC2 lo CD11b hi F4/80 lo ) in the livers of the indicated BM chimeras ( n = 5 or 6). The data are shown as the mean ± s.e.m. and were statistically analyzed by one-way ANOVA with Tukey’s multiple-comparison test ( e , l ) or two-tailed Student’s t -test ( g , h , i , j ). Data shown are representative of three independent experiments with similar results ( a – d ). Uncropped blots are provided in Source Data. All the p values were two-sided and adjustments were made for multiple comparisons. kD relative molecular weight in kilodalton, rA8 (or A8) recombinant ANGPTL8 protein, KCs Kupffer cells, MDMs monocyte-derived macrophages, PA palmitate, BM bone marrow, NS nonsignificant. Source data are available as a Source Data file.

Article Snippet: The PVDF membranes were blocked in blocking buffer for 2 h at RT, followed by incubation with the following antibodies overnight at 4 °C: mouse anti-β-Actin (Proteintech, Wuhan, China, Cat# 66009-1-Ig, 1:1000 dilution); rabbit anti-ANGPTL8 (Abcam, UK, Cat# ab180915, 1:1000 dilution), anti-ERK1/2 (Cell Signaling Technology, MA, USA, Cat# 4695T, 1:1000 dilution), anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, MA, USA, Cat# 4370T, 1:1000 dilution), anti-p38-MAPK (Cell Signaling Technology, MA, USA, Cat# 8690T, 1:1000 dilution), and anti-phospho-p38-MAPK (Thr180/Tyr182; Cell Signaling Technology, MA, USA, Cat# 4511T, 1:1000 dilution), anti-AKT (Cell Signaling Technology, MA, USA, Cat# 4691S, 1:1000 dilution), anti-phospho-AKT (Ser473; Cell Signaling Technology, MA, USA, Cat# 4060T, 1:1000 dilution), anti-NF-κB-p65 (Cell Signaling Technology, MA, USA, Cat# 8242T, 1:1000 dilution), anti-phospho- NF-κB-p65 (Ser536; Cell Signaling Technology, MA, USA, Cat# 3033T, 1:1000 dilution); mouse monoclonal anti-(His)6 antibody (Abcam, UK, Cat#ab237339, 1:2000 dilution); anti-SHP1 (Cell Signaling Technology, Shanghai, China, Cat# 3759, 1:1,000 dilution), anti-SHP2 (Cell Signaling Technology, Shanghai, China, Cat# 3397, 1:1000 dilution) and anti-phospho-tyrosine (Cell Signaling Technology, Shanghai, China, Cat# 9411 1:2000 dilution); anti-His (Abcam, UK, Cat# ab18184, 1:1000 dilution); anti-Flag (BioLegend, USA, # 637303, 1:500 dilution); and rat anti-PirB (Thermo Fisher Scientific, MA, USA, Cat# MA5-24049, 1:500 dilution).

Techniques: Translocation Assay, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay, Expressing, Migration, Flow Cytometry, Comparison, Two Tailed Test, Molecular Weight, Recombinant, Derivative Assay

Journal: Nature Communications

Article Title: LILRB2/PirB mediates macrophage recruitment in fibrogenesis of nonalcoholic steatohepatitis

doi: 10.1038/s41467-023-40183-3

Figure Lengend Snippet: a Immunocytochemistry staining of Flag-targeted ANGPTL8 and LILRB2 on human monocyte-derived macrophages (hMDMs) treated with exogenous rA8 (100 ng/ml). Scale bar, 10 µm. b LILRB2 mRNA expression in primary human peripheral blood monocytes from healthy adults and NASH patients ( n = 10). c Flow cytometry analysis (left) of LILRB2 in the blood Mos (CD14+) from the indicated individuals. Mean fluorescence intensity (MFI) (right) of LILRB2 in blood Mos (CD14+) from the indicated individuals ( n = 7). d Cytokine mRNA expression in hMDMs treated with scrambled protein and rA8 (n = 6). e Migration of CD14+ monocytes to ANGPTL8 were abrogated by the anti-LILRB2 antibody ( n = 10). f Western blot analysis of the phosphorylation of signaling molecules downstream of LILRB2 in hMDMs treated with rA8. Uncropped blots are provided in Source Data. g Migration of CD14+ monocytes pretreated with phosphorylation inhibitors before rA8 or scrambled protein stimulation. U0126 p-ERK1/2 inhibitor, SB203580 p-P38 inhibitor, MK-2206, p-AKT inhibitor, BAY11-7082 p-P65 inhibitor ( n = 4 or five independent experiments). (h) Experimental scheme to coculture hepatocytes with hMDMs. i , j Primary human hepatocytes were cocultured with monocyte-derived macrophages and treated with rA8 and PA, after which Oil Red O and TG ( n = 9 biologically independent samples) were measured (scale bar, 100 µm). The data are shown as the mean ± s.e.m. and were statistically analyzed by two-tailed Student’s t test ( b, c , g , j ) and one-way ANOVA with Tukey’s multiple-comparison test ( d , e ). All samples are biologically independent replicates and n indicates the number of biologically independent samples examined ( b – e ). Data shown are representative of three independent experiments with similar results ( a , f ). All the p values were two-sided and adjustments were made for multiple comparisons. kD relative molecular weight in kilodalton, hMDMs human monocyte-derived macrophages, Mo monocytes, rA8 recombinant ANGPTL8 protein, MFI mean fluorescence intensity. Source data are available as a Source Data file.

Article Snippet: The PVDF membranes were blocked in blocking buffer for 2 h at RT, followed by incubation with the following antibodies overnight at 4 °C: mouse anti-β-Actin (Proteintech, Wuhan, China, Cat# 66009-1-Ig, 1:1000 dilution); rabbit anti-ANGPTL8 (Abcam, UK, Cat# ab180915, 1:1000 dilution), anti-ERK1/2 (Cell Signaling Technology, MA, USA, Cat# 4695T, 1:1000 dilution), anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Technology, MA, USA, Cat# 4370T, 1:1000 dilution), anti-p38-MAPK (Cell Signaling Technology, MA, USA, Cat# 8690T, 1:1000 dilution), and anti-phospho-p38-MAPK (Thr180/Tyr182; Cell Signaling Technology, MA, USA, Cat# 4511T, 1:1000 dilution), anti-AKT (Cell Signaling Technology, MA, USA, Cat# 4691S, 1:1000 dilution), anti-phospho-AKT (Ser473; Cell Signaling Technology, MA, USA, Cat# 4060T, 1:1000 dilution), anti-NF-κB-p65 (Cell Signaling Technology, MA, USA, Cat# 8242T, 1:1000 dilution), anti-phospho- NF-κB-p65 (Ser536; Cell Signaling Technology, MA, USA, Cat# 3033T, 1:1000 dilution); mouse monoclonal anti-(His)6 antibody (Abcam, UK, Cat#ab237339, 1:2000 dilution); anti-SHP1 (Cell Signaling Technology, Shanghai, China, Cat# 3759, 1:1,000 dilution), anti-SHP2 (Cell Signaling Technology, Shanghai, China, Cat# 3397, 1:1000 dilution) and anti-phospho-tyrosine (Cell Signaling Technology, Shanghai, China, Cat# 9411 1:2000 dilution); anti-His (Abcam, UK, Cat# ab18184, 1:1000 dilution); anti-Flag (BioLegend, USA, # 637303, 1:500 dilution); and rat anti-PirB (Thermo Fisher Scientific, MA, USA, Cat# MA5-24049, 1:500 dilution).

Techniques: Immunocytochemistry, Staining, Derivative Assay, Expressing, Flow Cytometry, Fluorescence, Migration, Western Blot, Phospho-proteomics, Two Tailed Test, Comparison, Molecular Weight, Recombinant

Journal: Oncogene

Article Title: Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases.

doi: 10.1038/sj.onc.1209664

Figure Lengend Snippet: Figure 2 E-selectin-mediated adhesion of colon cancer HT-29 cells to HUVEC induces the activation of ERK and p38 MAP kinases in HUVEC – HUVEC were stimulated with 20 ng/ml IL-1b for 4 h. Thereafter, they were treated or not with E-selectin antibody (H18/7) for 30 min at 41C. Then, HT-29 cells were fixed for 20 min with paraformaldehyde 2%, and were seeded on HUVEC for 30 min at 41C. Then, the cultures were transferred at 371C for increasing periods of time, and ERK (a) and p38 (b) kinase activation was determined in Western blot using phosphos- pecific antibodies. Results are the mean7s.d. of three separate experiments. Representative autoradiograms are shown.

Article Snippet: Mouse monoclonal antiphospho ERK MAP kinase, rabbit polyclonal anti-phospho Myosin Light Chain 2, rabbit polyclonal anti-Myosin Light Chain 2, rabbit polyclonal anti-phospho p38 MAP kinase were purchased from Cell Signalling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Oncogene

Article Title: Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases.

doi: 10.1038/sj.onc.1209664

Figure Lengend Snippet: Figure 6 p38-mediated formation of stress fibres in response to activation of E-selectin – HUVEC were stimulated with 20 ng/ml IL- 1b for 4 h. Forty-five minutes before the end of treatments DMSO (a–f) or SB203580 (5 mM) (g–i) were added in the medium. Thereafter, HUVEC were (d–i) or not (a–c) exposed to the E-selectin activating antibody system as described in Figure 1. F-actin was detected by phalloidin isothiocyanate conjugated with fluorescein (b, c, e, f, h and i). Total E-selectin was detected using the monoclonal antibody (H18/7) (a, c, d, f, g, i). The antigen–antibody complex was detected by a goat anti-mouse antibody conjugated with Alexa-568. The cells were examined with the use of Nikon Eclipse E800 fluorescence equipped with a 40 objective. Representative fields are shown. Phosphorylation of MLC was determined in j and k. HUVEC were stimulated with 20 ng/ml IL-1b for 4 h. Forty-five minutes before the end of treatment DMSO or the following inhibitors were added in distinct wells: SB203580 (S; 5 mM), cytochalasin D (C; 50 nM), ML-7 (M; 25 mM). Thereafter, HUVEC were exposed or not to the E-selectin activating antibody system. Phosphorylation of MLC was determined in Western blot using a phosphospecific antibody. Results are the mean7s.d. of two separate experiments. Representative autoradiograms are shown. Arrows show E-selectin at cell–cell junctions.

Article Snippet: Mouse monoclonal antiphospho ERK MAP kinase, rabbit polyclonal anti-phospho Myosin Light Chain 2, rabbit polyclonal anti-Myosin Light Chain 2, rabbit polyclonal anti-phospho p38 MAP kinase were purchased from Cell Signalling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot