Journal: Lupus Science & Medicine

Article Title: Truncated isoforms of GPSM2 containing the GoLoco motif region promote CD4 + T-cell migration in SLE

doi: 10.1136/lupus-2022-000742

Figure Lengend Snippet: A truncated isoform of GPSM2 promotes CD4 + T-cell migration in SLE. (A) Representative example of western blot analysis of GPSM2 and the truncated 35 kDa GPSM2 isoform in CD4 + T cells from patients with SLE (SLE) and HCs. GPSM2 overexpressing lysate served as a positive ctrl (+); no protein served as a negative ctrl (−). (B) Comparison of GPSM2 expression in CD4 + cells from patients with SLE (n=10) and healthy individuals (n=9). (C) Expression of the truncated GPSM2 isoform in CD4 + cells from patients with SLE (n=10) and healthy individuals (n=9). (D) Schematic view of the experimental set-up of an in vitro T-cell migration assay. (E) Inhibition of the truncated GPSM2 isoform with blocking antibodies (block) or ctrl antibodies leads to decreased migration of CD4+ T cells from patients with SLE (n=9). Data are presented as mean±SEM. Significance was calculated using unpaired (B, C) or paired (E) Student’s t-test. ctrl, control; GPSM2, G-protein signalling modulator 2; HC, healthy control; n.s., not significant.

Article Snippet: GPSM2 overexpressing lysate served as a positive control (Novus Biologicals, Centennial, USA; cat. no. NBL1-11310).

Techniques: Migration, Western Blot, Comparison, Expressing, In Vitro, Cell Migration Assay, Inhibition, Blocking Assay, Control

Journal: Nucleic Acids Research

Article Title: CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

doi: 10.1093/nar/gkv434

Figure Lengend Snippet: DSB induced degradation of MDC1 happens more promptly in S-phase. ( A ) HEK 293T cells were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (5 Gy) and harvested at indicated time points. Left panel: the level of endogenous MDC1 was examined with indicated antibodies by immunoblotting. Blots were quantified using ImageJ (National Institutes of Health). Right panel: the cell cycle profiles of the cells are analyzed by flow cytometry. ( B ) U2OS cells were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (2 Gy) and probed with indicated antibodies at the indicated time point. Representative images are shown in the left panels, the quantification of the percentage of cells displaying MDC1 (upper) or Rad51 (lower) foci are shown in right panels. Error bars represent SEM. from three independent experiments. ** P < 0.01 two‐tailed Student's t ‐test. >200 cells were counted per experiment. ( C ) HEK 293T cells transfected with HA-MDC1 and FLAG-SUMO1 were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (5 Gy) and harvested at indicated time points. MDC1 was then immunoprecipitated with anti-HA antibody and MDC1 sumoylation was examined using anti-FLAG antibody. Blots were quantified using ImageJ (National Institutes of Health). ( D ) HEK 293T cells transfected with Myc-RNF4 were synchronized by double thymidine block or released into S phase. The cells were irradiated (5 Gy) and then collected at indicated time points. RNF4 was then immunoprecipitated and subjected to in vitro ubiquitination reactions in the presence of HA-Ub, UbcH5a and UBE1. The ubiquitination of RNF4 were analyzed by immunoblot as indicated.

Article Snippet: Sumoylated GST-MDC1(aa1818–2089) bound to GST-sepharose were washed with Tris-HCl (50 mM, pH7.5) and then incubated with 200 ng of UbcH5a (Boston Biochem), 300 ng of ubiquitin activating enzyme (UBE1) (Boston Biochem), 2 μg of HA-ubiquitin (Boston Biochem), 3 μg Myc-RNF4 or FLAG-RNF4 in 40 μl of reaction buffer (50 mM Tris[pH7.5], 2.5 mM MgCl 2 , 2 mM ATP, 2 mM DTT).

Techniques: Blocking Assay, Irradiation, Western Blot, Flow Cytometry, Two Tailed Test, Transfection, Immunoprecipitation, In Vitro, Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

doi: 10.1093/nar/gkv434

Figure Lengend Snippet: The phosphorylation on RNF4 affects its activity. ( A ) Upper panel: HEK 293T cells transfected with FLAG-RNF4 WT, 2TA or 2TE mutant were synchronized in G1 phase by double thymidine block or released into S phase. The cells were irradiated (5 Gy) and then collected at indicated time points. RNF4 was then immunoprecipitated and subjected to in vitro ubiquitination reactions in the presence of HA-Ub, UbcH5a and UBE1. The ubiquitination of RNF4 were analyzed by immunoblot as indicated. Lower panel: the cell cycle profiles of the cells were analyzed by flow cytometry. ( B ) Upper panel: schematic of the combined in vitro sumoylation and ubiquitination assay procedures. Lower panel: bacterially expressed GST‐MDC1 (aa 1818–2089) bound to GST‐sepharose were subjected to combined in vitro sumoylation and ubiquitination reactions as shown in the upper panel. The GST‐MDC1(aa 1818–2089) was subjected to in vitro sumoylation reactions in the presence of SUMO1 and then subjected to in vitro ubiquitination reactions in the presence or absence of HA‐Ub, RNF4‐WT, RNF4‐2TA, or RNF4-2TE mutant as indicated. The ubiquitination of GST‐sepharose‐bound protein were analyzed by immunoblot as indicated. ( C ) Cells depleted RNF4 with siRNA were cotransfected with HA-MDC1 and FLAG-RNF4-WT, 2TA or 2TE mutant. The cells were synchronized in G1 phase by double thymidine block or released into S phase and then irradiated (5 Gy). The ubiquitination of HA-MDC1 was then analyzed with indicated antibodies.

Article Snippet: Sumoylated GST-MDC1(aa1818–2089) bound to GST-sepharose were washed with Tris-HCl (50 mM, pH7.5) and then incubated with 200 ng of UbcH5a (Boston Biochem), 300 ng of ubiquitin activating enzyme (UBE1) (Boston Biochem), 2 μg of HA-ubiquitin (Boston Biochem), 3 μg Myc-RNF4 or FLAG-RNF4 in 40 μl of reaction buffer (50 mM Tris[pH7.5], 2.5 mM MgCl 2 , 2 mM ATP, 2 mM DTT).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Mutagenesis, Blocking Assay, Irradiation, Immunoprecipitation, In Vitro, Ubiquitin Proteomics, Western Blot, Flow Cytometry

Journal: ACS omega

Article Title: Magnetoreception of Photoactivated Cryptochrome 1 in Electrochemistry and Electron Transfer.

doi: 10.1021/acsomega.8b00645

Figure Lengend Snippet: Figure 2. (A) Cyclic voltammograms for the gold slide surface immobilized with CRY1 with and without blue light excitation in the absence of the magnetic field and with blue light excitation under different magnetic fields at the scan rate of 4 V s−1. (B) Time profiles for the surface concentration of immobilized CRY1 with blue light excitation at the scan rate of 4 V s−1 in the absence of the magnetic field.

Article Snippet: Ultraviolet−visible spectroscopy (UV−vis spectroscopy, Varian Cary 6000i) over the spectral range of 280−600 nm was used to investigate the absorbance properties of the photolyase-like CRY1 (Novus Biologicals, cat.# NBL1-09495, licensed from OriGene Technologies), and the CRY1 proteins were created in HEK293T cells, using plasmid ID RC207152 and based on accession number NM_004075.

Techniques: Concentration Assay

Journal: ACS omega

Article Title: Magnetoreception of Photoactivated Cryptochrome 1 in Electrochemistry and Electron Transfer.

doi: 10.1021/acsomega.8b00645

Figure Lengend Snippet: Figure 4. Proposed electrochemical reaction and electron transfer pathways of CRY1 immobilized at the gold electrode based on the photocycle scheme of CRYs and the magnetic sensitive radical pair mechanism.

Article Snippet: Ultraviolet−visible spectroscopy (UV−vis spectroscopy, Varian Cary 6000i) over the spectral range of 280−600 nm was used to investigate the absorbance properties of the photolyase-like CRY1 (Novus Biologicals, cat.# NBL1-09495, licensed from OriGene Technologies), and the CRY1 proteins were created in HEK293T cells, using plasmid ID RC207152 and based on accession number NM_004075.

Techniques:

Journal: ACS omega

Article Title: Magnetoreception of Photoactivated Cryptochrome 1 in Electrochemistry and Electron Transfer.

doi: 10.1021/acsomega.8b00645

Figure Lengend Snippet: Figure 5. Magnetic sensitivity of (A) peak current (at 4 V s−1 scan rate) vs field strength and (B) plot of ln(km/k0) vs field strength for the gold slide surface immobilized with CRY1 under blue light excitation with a linear fit. Note that km and k0 are the calculated rate constant k0 values in the presence and absence of magnetic fields, respectively.

Article Snippet: Ultraviolet−visible spectroscopy (UV−vis spectroscopy, Varian Cary 6000i) over the spectral range of 280−600 nm was used to investigate the absorbance properties of the photolyase-like CRY1 (Novus Biologicals, cat.# NBL1-09495, licensed from OriGene Technologies), and the CRY1 proteins were created in HEK293T cells, using plasmid ID RC207152 and based on accession number NM_004075.

Techniques:

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: mRNA quantitative expression versus immunohistochemistry. (A) DCSIGN, CD163 M2, α‐smooth muscle actin (α‐SMA), fibroblast‐specific protein 1 (FSP1) and fibroblast activation protein (FAP) immunohistochemistry. Representative pictures of high and low protein expression levels of DCSIGN, CD163 M2 macrophage and α‐SMA, FSP1 and FAP cancer‐associated fibroblast (CAF) markers in human tumor samples. Arrows indicate positive cells. Original magnification, ×20. (B) Association between protein and mRNA expression levels. Statistical association between protein and mRNA expression levels of FSP1 and FAP, CAF markers. In addition, a clear trend toward statistical association is observed between protein and mRNA expression levels of DCSIGN and CD163, M2 macrophage markers and the α‐SMA, CAF marker. Bar charts show the average (±confidence interval) of mRNA expression levels of each gene in the low and high protein expression groups.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Marker

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and disease‐free survival (DFS) in colorectal cancer patients. Association between CD163, a M2 macrophage marker (A), and α‐smooth muscle actin (α‐SMA) (B), fibroblast‐specific protein 1 (FSP1) (C) and fibroblast activation protein (FAP) (D) CAF markers with DFS. “Low expression” refers to a low‐expression tertile for α‐SMA and low‐ and medium‐expression tertiles for CD163, FSP1 and FAP.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and overall survival in colorectal cancer patients. Association of CD163, a M2 macrophage marker (A), and fibroblast‐specific protein 1 (FSP1) (C), a CAF marker, with overall survival. α‐Smooth muscle actin (α‐SMA) (B) and fibroblast activation protein (FAP) (D) expression levels, CAF markers, showed a trend towards an association with overall survival.

Article Snippet: Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Experimental eye research

Article Title: Retinal gliosis and phenotypic diversity of intermediate filament induction and remodeling upon acoustic blast overpressure (ABO) exposure to the rat eye.

doi: 10.1016/j.exer.2023.109585

Figure Lengend Snippet: Fig. 4. Altered expression of retinal Type III IF desmin following single ABO exposure. Upper panels: A. Representative images of non-exposed control retinal desmin immunolabeling from nasal control and 1 wk post-ABO retinas. Left, Control, Nasal retina; GFAP-IF (red) was restricted largely to the astroglia/ILM, (upward blue arrows), while desmin (green) was constitutively expressed through Müller glial processes (green arrows). Desmin expression through OPL processes (cyan arrows) and vascular pericytes (asterisk) was observed. Right,1 wk post-ABO Nasal retina; Radial GFAP upregulation at 1 wk ABO without overt constitutive radial desmin remodeling was observed, with graded Müller cell co-expression of both IFs (yellow arrows); GFAP often extended into the outer retina without accompanying desmin IF (red arrows). While no overt changes in radial glial desmin were observed post-ABO, IF-remodeling through the ILM was observed, with greater lateral GFAP+/ desmin+ overlap through the inner retinal cytoskeletal structures (upward blue arrows). B. jONH Control retina (left) and 1 wk post-ABO (right) exhibited a similar GFAP/desmin co-immunolabeling pattern, with discrete lateral spreading of inner retinal IF post-exposure compared to controls. Lower panels: C,E. Western blot analyses of whole retina lysates probed using desmin-pAb following preabsorption against full-length vimentin and GFAP proteins. Sample sizes shown above bars. Immunoreactive doublets at 64/65 kDa, 54/55 kDa, and 47/48 kDa were observed (black arrows). 55 KDa is consistent with the predicted unmodified molecular weight of desmin. A lower-migrating species at Mr ~48 kDa could correspond to calpain-protease degradation product from desmin processing which migrates ~10 kDa lower than full-length desmin (Aweida et al., 2018). While no overall changes in desmin levels were seen at 48 h post-ABO, an increase in the levels of 54/55 kDa and 47/48 kDa desmin forms was observed at 1 wk post-ABO (E). D,F. Quantification of the major immunoreactive desmin forms confirmed no overall changes in retinal desmin protein levels at 48 h post-ABO. Quantification of 1 wk ABO lysates revealed elevated levels of 54/55 kDa, and 47/48 kDa desmin forms, consistent with the expected non-modified and lightly degraded desmin forms, respectively, and no significant change in 64/65 kDa species. G. Western blot analyses of Calpain 1 (Cal. 1) digested overexpressed-desmin and rat retina lysates probed with desmin pAb. 200 ng desmin overexpression lysate or 10 μg rat retina lysate was digested with 2 U calpain 1 with or without the presence of 2 mM protease inhibitor EGTA. Desmin pAb detected multiple MW protein forms ranging from 25 to 75 kDa in HEK293T desmin OE-lysate, absent in vector-control HEK293T lysates, and similar endogenous desmin expression patterns in rat retina lysates, with Cal. 1, resulting in collapse of the immunoreactive desmin forms between 25 and 35 kDa in both overexpression and rat retina lysates. Fully protected retina lysates showed even higher range of desmin species, showing that digest conditions (30 ◦C, without protease inhibitor cocktail, P8340), also led to minimal desmin degradation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Western blot analysis was performed, probing parallel blots of 10 ng human GFAPGST (N-terminally tagged) fusion protein (H00002670-P01, Novus Biologicals), 10 ng recombinant human vimentin (NBP2-35139, Novus Biologicals) or 10 μg (total protein) of desmin overexpression (OE) lysate (HEK293 T-antigen driven full-length human desmin, NBL109840, Novus Biologicals), using both 10 μg matched empty-vector HEK293T lysate and 10 ng protease-free BSA (A3059, MilliporeSigma, St Louis, MO) as negative controls.

Techniques: Expressing, Control, Immunolabeling, Western Blot, Molecular Weight, Modification, Over Expression, Protease Inhibitor, Plasmid Preparation

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Antibodies used for immunohistochemical staining of breast cancer tissue samples.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Immunohistochemical staining, Staining, Positive Control

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Positive control (a) and isotype control (b). For positive control tissues certainly expressing the questionable antigens were stained. Placental tissue was used for GALNT6 staining, Colon tissue was used for GCNT2 and Uterus was chosen for ST6GALNac1 staining. Furthermore appropriate antibody concentrations were determined in the positive controls. For isotype control same tissues as for positive control are used, but primary antibody is replaced by a control serum, thus excluding unspecific binding signals of the primary antibody.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Positive Control, Expressing, Staining, Binding Assay

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Staining of malignant breast tissue with antibodies against the three glycosyltransferases GALNT6 (a), GCNT2 (b), and ST6GALNac1 (c). Pictures were taken with different objectives (6,3x, 10x, 25x, and 40x; from left to right column) resulting in different magnifications of tissue structures.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Staining

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Statistical analysis of the investigated features. Statistically significant P values are seen for Her4 (all glycosyltransferases) and for tumour size (GALNT6; GCNT2 and ST6GALNac1 show borderline significance).

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: