Journal: Cell Death & Disease

Article Title: Naa10p promotes cell invasiveness of esophageal cancer by coordinating the c-Myc and PAI1 regulatory axis

doi: 10.1038/s41419-022-05441-0

Figure Lengend Snippet: A Stably Naa10p-V5-expressing KYSE70 cells were immunoprecipitated with an V5 antibody and blotted with anti-PAI1 and anti-Cystatin E/M antibody. B KYSE70 cells were immunoprecipitated with Naa15 antibody and blotted with anti-Naa10p and anti-PAI1 antibody. C KYSE70 cells with stable Naa10p knockdown were transfected with either Naa10p-V5 or Naa10p-R82A-V5. Cell extracts were immunoprecipitated with an anti-PAI1 antibody or anti-acetylated lysine antibody and blotted with anti-acetylated lysine and anti-PAI1 antibody. D KYSE70 cells with stable Naa10p knockdown were transfected with either Naa10p-V5 or Naa10p-R82A-V5. Cell extracts and conditioned medium of these cells were collected and immunoblotting analysis was performed. E Cell extracts and conditioned medium of KYSE70 cells receiving sh NAA15 or control shRNA were collected and immunoblotting analysis was performed. F Immunoblotting analysis of stably c-Myc-silencing KYSE70 cells transfected with either Ctl-shRNA or SERPINE1 shRNAs. G Quantitative data comparing cell invasion of stably c-Myc-silencing KYSE70 cells transfected with either Ctl-shRNA or SERPINE1 shRNAs. Bars are the mean ± SD of three independent experiments. ** P < 0.01 when compared to shCtl group and ## P < 0.01 when compared with sh MYC -shCtl cells by two-tailed Student’s t test. Scale bar: 100 μm. H Kaplan–Meier plot of overall (OS) survival of ESCA patients stratified by NAA10 and SERPINE1 expression level. A log rank test was used to show differences between groups.

Article Snippet: The following antibodies were used: Naa10p (GB-10511, Genesis biotech, Taiwan) for immunoblotting, c-Myc (ab32072, Abcam) for immunoblotting, Naa15 (sc365931, Santa Cruz) for immunoblotting and immunoprecipitation, Cystatin E/M (AF-1286, R&D) for immunoblotting, PAI1 (AF-1786, R&D) for immunoblotting, PAI1 (ab222754, Abcam) for immunoprecipitation, TIMP2 (AF-971, R&D) for immunoblotting, V5 (ab9116, Abcam) for immunoblotting and immunoprecipitation, Acetylated lysine (9681, Cell Signaling) for immunoblotting, Acetylated lysine (05-515, Sigma) for immunoprecipitation.

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Transfection, Western Blot, shRNA, Two Tailed Test

Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Increased intracellular latent TGFβ1 in ML-II fibroblasts is localized within dense, detergent-insoluble lysosomal fractions. ( A ) Western blot analysis of pooled Percoll gradient fractions from control and ML-II cellular lysates (n = 4). SDS-PAGE-resolved fractions were subjected to analysis using antibodies against markers for lysosomes (LAMP-2), Golgi (GS28) and ER (ERp29) as well as LAP. Pool I, dense membranes (lysosomes); pool II, intermediate density organelles; pool III, ER and Golgi membranes. ( B ) Western blots of human control and ML-II cells fractionated into deoxycholate (DOC)-soluble (S) and –insoluble (I) fractions (n = 4). Along with concentrated media (M) fractions, these pools were resolved by non-reducing (NR) SDS-PAGE and immunoblotted with antibodies against human LAP or LTBP-1. The upper and lower arrowheads indicate the position of the LLC and free LTBP-1, respectively ( C ) Luciferase assays (n = 3) of heat-treated deoxycholate-insoluble fractions that were applied to mink lung TGFβ1 reporter cells; luciferase activity was measured in relative luminescence units, ** p ≤ 0.01.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, SDS Page, Luciferase, Activity Assay

Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Impaired secretion and increased insolubility and of latent TGFβ1 and reduced TGFβ1 signaling, are also noted in feline ML-II fibroblast-like synoviocytes. ( A ) Western blots of feline control and ML-II cells fractionated into deoxycholate-soluble (S) and –insoluble (I) fractions (n = 3). These fractions and concentrated media (M) from cultures, were resolved by non-reducing (NR) SDS-PAGE and immunoblotted with an antibody against human LAP. ( B ) Western blot analysis of control and ML-II feline fibroblast lysates using antibodies against Smad 2/3 and phosphorylated Smad 2. ( C ) Quantification of the relative intensity of phosphorylated Smad 2 vs. Smad 2/3 protein in control and ML-II cells (n = 3), ** p ≤ 0.01.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, SDS Page

Journal: Biomolecules

Article Title: Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFβ in Mucolipidosis II Cells

doi: 10.3390/biom10050670

Figure Lengend Snippet: Sortilin-1 protein and transcript levels are elevated in ML-II but not ML-III, fibroblasts. Western blots for sortilin-1 in control and ML-II human fibroblasts ( A ) and feline fibroblast-like synoviocytes ( B ) using a mouse monoclonal antibody against human sortilin-1 (n = 3). ( C ) Sortilin-1 Western blot in WT and GNPTAB-/- (KO) HeLa cells (n = 2). ( D ) Sortilin-1 Western blot of DOC-soluble and –insoluble fractions of human control and ML-II fibroblasts (n = 3). ( E ) Western blot of control and ML-II feline chondrocytes (FC) and fibroblast-like synoviocytes (FLS) as well as control and ML-II human fibroblasts (HF) using a polyclonal antibody against human cathepsin D (n = 2). ( F ) Immunoblots for sortilin-1 and LAP in control, ML-III and ML-II human fibroblasts. Note the correlation of increased sortilin levels and accumulation of LAP in ML-II but not ML-III cells. ( G ) Representative reverse transcription-polymerase chain reaction (RT-PCR) analysis of sortilin-1 transcripts. Ribosomal Protein L4 (RPL4) transcript abundance is shown as a loading control. ( H ) Quantification of the intensity of SORT1 relative to RPL4 (n = 3), *** p ≤ 0.001.

Article Snippet: Goat antisera against human LAP and monoclonal antibody against human LTBP was purchased from R & D systems (USA), while the monoclonal antibody γ-tubulin was obtained from Sigma.

Techniques: Western Blot, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: EBioMedicine

Article Title: Autocrine CTHRC1 activates hepatic stellate cells and promotes liver fibrosis by activating TGF-β signaling

doi: 10.1016/j.ebiom.2019.01.009

Figure Lengend Snippet: CTHRC1 activates both TGF-β and Wnt signaling, while the promotive effect of CTHRC1 on HSC activation is mainly dependent on TGF-β signaling. A. The expression of α-SMA in primary HSCs after 7 days isolated from WT and CTHRC1 −/− mice. B. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein alone, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG. C. Western blotting analysis of phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in five liver tissues of WT or CTHRC1 −/− mice intraperitoneally injected with CCl 4 . GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. D. Phosphorylation of Smad2, Smad3, JNK, total Smad4 and expression of Wnt5a in primary rat HSCs, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus CTHRC1 mAb or IgG for 1, 3, 5 days, individually. GAPDH was the loading control. The densitometry of p-Smad2/Smad2 was shown below. E and F. Representative immunofluorescence images of α-SMA (green in E, red in F) in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus neutralizing antibodies or inhibitor as follows: TGFBR2 neutralizing antibody or TGF-β receptor inhibitor (E), Wnt5a or Wnt3a neutralizing antibody (F). Nuclei are stained with DAPI (blue). Scale bars, 50 μm. G. The expression of α-SMA in primary rat HSCs after 4 days, which were treated with vehicle, 20 nM rCTHRC1 protein, and 20 nM rCTHRC1 protein plus TGFBR2 neutralizing antibody or TGF-β receptor inhibitor. GAPDH was the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Recombinant CTHRC1 and/or TGFBR2 neutralizing antibody (R&D, AF-241-NA), TGF-βR inhibitor, LY2109761 (SELLECK, S2704) were added in the medium simultaneously.

Techniques: Activation Assay, Expressing, Isolation, Western Blot, Injection, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus

doi: 10.3389/fimmu.2019.03131

Figure Lengend Snippet: Identification of the neuron, astrocyte, and monocytes/macrophages (MM). (A) The PBMCs were isolated from the duck whole blood cells (WBCs); both cells were stained with Wright Strain. At least 15 individual views were observed to calculate the cell populations. (B) The time course of PBMCs differentiates into monocytes/macrophages. (C–E) Three duck primary cell types were identified via IFA using a rabbit polyclonal antibody against MAP2 for neurons (C) , a rabbit polyclonal antibody against GFAP for astrocytes (D) , and a mouse polyclonal antibody against CD80 and CD86 for monocytes/macrophages (E) . The scale bar is 100 μm. (F) The monocytes/macrophages or DEF cells were lysed with RIPA buffer, and the protein expression levels were determined by Western blot using antibodies against CD80, CD80, or β-actin. (G) The basal levels of duck TLR2, TLR4, CD86, and CD80 in the five types of duck primary cells were examined using Q-PCR with specific primers. The data are presented as Cq values for 100 ng of RNA.

Article Snippet: The Rabbit anti-human MAP2 and -human GFAP polyclonal antibody and the goat anti-human β-actin monoclonal antibody were purchased from Abclonal (Wuhan, China).

Techniques: Isolation, Staining, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus

doi: 10.3389/fimmu.2019.03131

Figure Lengend Snippet: Blockage of IFNAR signaling enhanced replication of the attenuated DPV strain (CHa) in astrocytes, PBMCs, and monocytes/macrophages. (A–C) DEFs were pretreated with the IFNAR inhibitor ruxolitinib at a concentration of 5 μM for 1 h and then infected with DTMUV at a MOI of 0.1. After infection, the inhibitor was kept at the same concentration for 24 h. The expression levels of IFN-β, MX, and IL-6 were determined by Q-PCR at 24 h post-infection. The data are presented as the ratio to β-actin. (D) The DEF cell was treated with ruxolitinib at a dose of 0.5, 1, 5, 10, and 20 μM/ml for 24 h; DMSO was added in the mock group for control, and then the cell viability was tested using MTT method. The results were presented as percentages (%) to mock. (E,F) Duck DEFs, neurons, astrocytes, PBMCs, and monocytes/macrophages were pretreated with ruxolitinib at a concentration of 5 μM/ml for 1 h and then infected with either the CHv or CHa strain at a MOI of 0.1. After infection, the inhibitor was kept at the same concentration. The cell culture supernatants were collected at 24 and 48 h post-infection, and the viral titers were determined in the cell culture supernatant based on the TCID 50 .

Article Snippet: The Rabbit anti-human MAP2 and -human GFAP polyclonal antibody and the goat anti-human β-actin monoclonal antibody were purchased from Abclonal (Wuhan, China).

Techniques: Concentration Assay, Infection, Expressing, Control, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury

doi: 10.1186/s12974-021-02283-z

Figure Lengend Snippet: Antibodies used in the present study

Article Snippet: Anti-activating transcription factor 3 (ATF3) (a marker of damaged neuron) , HPA001562 , Rabbit , Polyclonal , 1:1000 , Atlas Antibodies, Stockholm, Sweden , AB_1078233.

Techniques: Concentration Assay, Marker, Binding Assay, Plasmid Preparation

Journal: Journal of Neuroinflammation

Article Title: Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury

doi: 10.1186/s12974-021-02283-z

Figure Lengend Snippet: Ganglionic macrophages proliferate after nerve injury. ATF3-positive cells (green) in the contralateral (contra) and ipsilateral (ipsi) sides of the maxillary nerve region of the trigeminal ganglion on day 1 after infraorbital nerve ligation ( a ) and the number of ATF3-positive cells ( n = 4–6/timepoints) ( b ). Iba1-positive cells (red) on day 7 after nerve ligation ( c ) and the number of Iba1-positive cells ( n = 4–6/timepoints) ( d ). BrdU (green)- and Iba1 (red)-positive cells ( e ), multiple staining showing co-localization (arrowhead) of BrdU signals (green) with nucleus (blue) of Iba1-positive cells (red) ( f ) on day 5 after a nerve ligation, and the number of BrdU- and Iba1-positive cells ( n = 4–6/timepoints) ( g ). See list of abbreviations. Scale bars are indicated. Data are represented as mean (S.D.), and differences were detected using two-way ANOVA with Tukey–Kramer test ( b , d , g )

Article Snippet: Anti-activating transcription factor 3 (ATF3) (a marker of damaged neuron) , HPA001562 , Rabbit , Polyclonal , 1:1000 , Atlas Antibodies, Stockholm, Sweden , AB_1078233.

Techniques: Ligation, Staining

Journal: Journal of Neuroinflammation

Article Title: Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury

doi: 10.1186/s12974-021-02283-z

Figure Lengend Snippet: Volume of ganglionic macrophages enlarges after nerve injury. Iba1-positive cells (red) in the contralateral (contra) and ipsilateral (ipsi) sides of the maxillary nerve region of the trigeminal ganglion on day 7 after infraorbital nerve ligation ( a ) and the cell areas of Iba1-positive cells ( n = 4–6/timepoints) ( b ). Z-stack images showing Iba1-positive cells (red) around ATF3-positive cells (green) ( c ), three-dimensional images showing Iba1-positive cells ( d ), and the cell volume of Iba1-positive cells ( n = 10 cells/group from 3 mice) ( e ) on day 7 after nerve ligation. See list of abbreviations. Scale bars are indicated. Data are represented as mean (S.D.), and differences were detected using two-way ANOVA with Tukey–Kramer test ( b ) and Student’s t test with Welch’s correction ( e )

Article Snippet: Anti-activating transcription factor 3 (ATF3) (a marker of damaged neuron) , HPA001562 , Rabbit , Polyclonal , 1:1000 , Atlas Antibodies, Stockholm, Sweden , AB_1078233.

Techniques: Ligation

Journal: Journal of Neuroinflammation

Article Title: Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury

doi: 10.1186/s12974-021-02283-z

Figure Lengend Snippet: Contact areas of ganglionic macrophages to primary sensory neurons are expanded after nerve injury. ATF3 (green)-, Iba1 (red)-, and PGP9.5 (blue)-positive cells in ipsilateral (ipsi) sides of the maxillary nerve region of the trigeminal ganglion on days 1 and 7 after infraorbital nerve ligation ( a ) and a percentage of contact-like structures (CLS) between ATF3-positive or negative neurons and Iba1-positive cells ( n = 4 or 5/group) ( b ). Multiple staining ( c ) and Z-stack images ( d ) showing Iba1-positive cells (green), Nissl-positive neurons (blue), and glutamine synthetase (GS)-positive satellite glial cells (red) in the contralateral (contra) and ipsilateral sides on day 7 after nerve ligation. White arrowhead indicates the contact sites between Iba1-positive cells and neurons. Three-dimensional images showing contact area of Iba1-positive cells and Nissl-positive neurons ( e ). Blue indicates the surface of Nissl-positive neurons, and yellow indicates the surface of Nissl-positive neurons with Iba1-positive cells in contact. Surface area of Iba1-positive or GS-positive satellite glial cells in contact with individual Nissl-positive neurons ( f ) and a percentage of these areas averaged together (averaging 10 cells/ipsi from 3 mice) ( g ) on day 7 after nerve ligation. Electron micrograph showing macrophages with electron-dense lipid bodies (asterisk) and lysosomes (arrow) in contact (black arrowhead) with neurons ( h ). “N” indicates the nucleus. See list of abbreviations. Scale bars are indicated

Article Snippet: Anti-activating transcription factor 3 (ATF3) (a marker of damaged neuron) , HPA001562 , Rabbit , Polyclonal , 1:1000 , Atlas Antibodies, Stockholm, Sweden , AB_1078233.

Techniques: Ligation, Staining

Journal: Frontiers in Immunology

Article Title: TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

doi: 10.3389/fimmu.2021.672143

Figure Lengend Snippet: TLR9 agonist CpG-B promotes IgG responses in humanized mice vaccinated with CD40-targeting vaccine. Humanized mice were treated with PBS control (n=3) or vaccinated with αCD40-HIV5pep (n=3) alone or vaccinated with αCD40-HIV5pep plus CpG-B (n=4) at week0, week3 and week6. At week 7, mice were sacrificed. (A, B) The expression of IgM and IgG on B cells from PBMCs and spleens was detected by FACS. (C) The total level of IgM and IgG in the plasma was detected by ELISA. (D) Splenocyte from humanized mice were cultured ex vivo in the present of R848(1μg/ml) and IL-2(10 u/ml) for 48hours, the cells were used for total IgG detection by ELISpot. (E) Antigen specific IgG level in the plasma was detected by ELISA. (F) The expression of activation-induced cytidine deaminase (AID) in spleen cells was detected by RT-PCR. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Article Snippet: Total IgM and IgG were detected by ELISA kits, purchased from Bethyl Laboratories,int.(Cat.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunospot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CPEB3 inhibits translation of mRNA targets by localizing them to P bodies

doi: 10.1073/pnas.1815275116

Figure Lengend Snippet: Antibodies

Article Snippet: Coverslips were mounted using FluorSave Reagent (Calbiochem) and images were acquired on an Olympus IX81 laser-scanning confocal microscope using the FluoView FV1000 Microscopy System. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antigen Species WB IF IP Ago2 (2A8) Mouse 1:1,000 1:400 – CPEB3 (Abcam) Rabbit 1:1,000 1:100 – CPEB3 (08B1) homemade Rabbit 1:2,000 1:200 1:20 HA (Covance) Mouse 1:1,000 1:1,000 1:25 GFP (Clontech) Mouse – – 1:25 GFP (G1) Mouse 1:200 – – GW182 (4B6) Mouse 1:1,000 1:100 2 μg Anti-mouse HRP (Santa Cruz) Donkey 1:10,000 – – Anti-rabbit HRP (Santa Cruz) Donkey 1:10,000 – – Anti-mouse/rabbit 700 (LI-COR) Donkey 1:15,000 – – Anti-mouse/rabbit 800 (LI-COR) Donkey 1:15,000 – – Anti-mouse/rabbit Cy3 (Jackson Laboratory) Goat – 1:500 – Anti-mouse/rabbit Cy5 (Jackson Laboratory) Goat – 1:500 – Pumilio 1 (A300-201A; Bethyl) Goat – 1:1,000 TIA-1 (Santa Cruz) Goat 1:1,000 1:100 CCR4 (Santa Cruz) Rabbit 1:1,000 1:200 FMRP (Millipore) Mouse 1:2,000 1:200 SUMO1 (Invitrogen) Mouse 6 μL/mL – 5 μg SUMO2 (Invitrogen) Rabbit 1/500 – 5 μg SUMO3 (Life Technologies) Rabbit 6 μL/mL – 5 μg Open in a separate window IF, immunofluorescence; IP, immunoprecipitation; WB, Western blotting.

Techniques: