Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a , b EAF1 C-terminal region and MED26 N-terminal region are required for CB–HLB association. Wild-type cells and EAF1-mutant cells ( a ) or MED26-mutant cells ( b ) were fixed with paraformaldehyde, and immunofluorescence staining for Coilin and NPAT was performed. Scale bar, 5 μm. Enlarged images for representative particles are shown in the lower part of each image. Evaluation of CB–HLB association in each mutant cell line is shown in the right panels. The NPAT particles were extracted from images. The area of NPAT particles occupied by Coilin particles and the intensity of the particles of NPAT and Coilin were calculated. The density shows the degree to which dots overlap with others. Signal intensities were evaluated using more than 200 nuclei for each condition. Wild-type, n = 308; EAF1 mutant, n = 204 in a , and wild-type, n = 278; MED26 mutant, n = 318 in b . c , d Quantification of the number and intensity of Coilin particles. All detected Coilin particles in EAF1-mutant cells (EAF1-mutant) ( c ) or MED26-mutant cells (MED26-mutant) ( d ) are plotted. NPAT-associating Coilin particles are indicated by black dots and free Coilin particles are indicated by white dots. Quantification was performed using 300 ( c ) or 200 ( d ) Coilin particles. e Super-resolution images showing relative positions of HLBs, Mediator, and CBs. Wild-type cells (WT, upper) and EAF1-mutant cells (EAF1 mut, lower) were fixed with paraformaldehyde, and triple immunofluorescence staining for NPAT (green), MED26 (red), and Coilin (gray) was performed. Enlarged images for representative particles are shown in the lower part of each image. f Calculation of the distance between HLBs and CBs. Immunofluorescence staining for NPAT and Coilin was performed with wild-type and EAF1-mutant HEK293T cells. For Coilin-associated NPAT particles, the distance between the center of the NPAT particle and Coilin particle was measured, and the distance between each associating particle is displayed as boxplots. The center line of each boxplot represents the median. Upper and lower fences of each boxplot represent upper and lower quartiles, respectively. Wild-type, n = 465 particles from 301 nuclei; EAF1 mutant, n = 145 particles from 354 nuclei. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Mutagenesis, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a – f HeLa cells were fixed with paraformaldehyde and subjected to triple immunofluorescence staining. MED26 (red), MED1 (red), ICE1 (red), ELL (red), LSM11 (red), and FLASH (red) were co-stained with NPAT (green) and Coilin (gray) using specific antibodies. Scale bar, 5 μm. Averaged signals of immunofluorescence centered at NPAT signal and respective line plots for each immunofluorescence experiment are shown on the right. Scale bar, 1 μm. g , h HCT116 cells were stained with anti-MED26 (green), anti-NPAT (green), and anti-Coilin (magenta) antibodies, and the nuclear positions of MED26 (Mediator), NPAT (HLBs), and Coilin (CBs) were observed by stimulated emission depletion (STED) microscopy. Scale bar, 1 μm. Line intensity plots across the particles of NPAT (green), Coilin (magenta), and MED26 (magenta) are shown in the lower panels. i Ratios of ICE1, ELL, LSM11, MED1, MED26, or FLASH colocalization with CBs, HLBs, or both CBs and HLBs. The ratio was calculated using more than 100 nuclei. ICE1, n = 71 particles from 196 nuclei; ELL, n = 122 particles from 240 nuclei; LSM11, n = 161 particles from 242 nuclei; MED1, n = 84 particles from 237 nuclei; MED26, n = 83 particles from 204 nuclei; FLASH, n = 187 particles from 291 nuclei. Source data are provided as a Source Data file. j Model of the relative position of each protein at CBs and HLBs. Mediator was located between HLBs and CBs.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Immunofluorescence, Staining, Microscopy

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a Schematic illustration of the strategy for in situ biotinylation of nuclear bodies. Cells were fixed and permeabilized, and HRP-conjugated antibodies were deposited onto target proteins (Coilin or LSM11). Then, the proximal chromatin was biotinylated by adding biotin–phenol and H 2 O 2 . After cell lysis, biotinylated proteins and their associated DNA were avidin-purified, followed by qPCR or high-throughput DNA sequencing. b – d CBs were dissociated from RDH gene clusters in EAF1-mutant cells. b Heatmap showing the CB (Coilin)-association profile in wild-type HEK293T cells. The avidin-purified DNA was analyzed by high-throughput sequencing. Two RDH gene clusters located at chromosome 1 and chromosome 6 are indicated by arrowheads. c Genome browser tracks showing the distribution of in situ biotinylation-seq reads using anti-Coilin antibodies at RDH gene cluster 2 in wild-type (WT) and EAF1-mutant (mut) cells. d Total counts of Coilin in situ biotinylation-seq reads at RDH genes, snRNA genes and snoRNA genes, and CB enrichment levels were compared between wild-type (WT) and EAF1-mutant (EAF1 mut) cells. Each value is the mean of two independent experiments. Source data are provided as a Source Data file. e Genome browser tracks showing the distribution of in situ biotinylation-seq using anti-Coilin antibodies at snRNA gene RNU2 in wild-type (WT) and EAF1-mutant (mut) cells. f Heatmap showing the U7 snRNP (LSM11)-association profile in wild-type HEK293T cells. The avidin-purified DNA was analyzed by high-throughput sequencing. Two RDH gene clusters located at chromosome 1 and chromosome 6 are indicated by arrowheads. g Genome browser tracks showing the distribution of in situ biotinylation-seq reads using anti-LSM11 antibodies at RDH gene cluster 2 in wild-type (WT) and EAF1-mutant (mut) cells. h Heatmap showing CB enrichment and mRNA expression levels of RDH genes in wild-type (WT) HEK293T cells, and fold-change of CB enrichment and mRNA expression levels of RDH genes in EAF1-mutant (EAF1 mut) cells. i Immunofluorescence images showing CDK11 localization at CBs. HCT116 cells were fixed with methanol and subjected to immunofluorescence staining. CDK11 (green) and Coilin (red) were stained using specific antibodies. Scale bar, 10 μm. j Meta-gene analysis of PRO-seq reads around TESs. Coilin (light blue) and CDK11 (yellow) iCLIP data by Machyna et al. and Sathyan et al. are overlaid with PRO-seq data. Approximate positions of stem-loop (SL) and HDE are indicated in the lower panel.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: In Situ, Lysis, Avidin-Biotin Assay, Purification, High Throughput Screening Assay, DNA Sequencing, Mutagenesis, Next-Generation Sequencing, Expressing, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

doi: 10.1038/s41467-022-30632-w

Figure Lengend Snippet: a Wild-type HEK293T (WT) and EAF1-mutant cells (mut) were fixed with paraformaldehyde containing 0.5% Triton X-100 and subjected to DNA-FISH. Four-color microscopy images are shown. RDH gene clusters 1 (cyan) and 2 (red) were stained using Cy5- or Cy3-labeled probe. NPAT (blue) and Coilin (green) were stained using specific antibodies. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 10 μm. The frequencies of each RDH gene cluster 2 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the left panel. The frequencies of each RDH gene cluster 1 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the right panel. b Representative four-color microscopy images showing the overlap of CB, HLB, and two RDH gene clusters in wild-type HEK293T cells. RDH gene clusters, NPAT, and Coilin were stained as described above. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 5 μm. The frequency of inter-chromosome interaction between two RDH gene clusters is shown in the right panel (WT, n = 1130 RDH gene cluster 2; mut, n = 771 RDH gene cluster 2). c – f 4C-seq analysis revealed that the higher-order inter-chromosome structure between two RDH gene clusters was abolished in EAF1-mutant cells. c , e Representative 4C-seq profile using RDH gene cluster 1 ( HIST1H2BK , chromosome 6) bait or RDH gene cluster 2 ( HIST2H2AB , chromosome 1) bait. The detailed contact profile at RDH gene cluster 2 (chromosome 1) ( c ) or at RDH gene cluster 1 (chromosome 6) ( e ) is shown in the lower panels. d , f Comparisons of 4C-seq counts detected in each RDH gene cluster region in wild-type (WT) or EAF1-mutant (EAF1 mut) cells under each 4C-seq condition. The read counts of 4C-seq using chromosome 6 HIST1H2BK bait ( d ) and chromosome 1 HIST2H2AB bait ( f ) are shown. These plots represent the mean of n = 2 experiments. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: anti-EAF1 antibodies (1:200 dilution, sc-398450; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MED26 antibodies (D4B1X, 1:1000 dilution, 14950; Cell Signaling Technology, Danvers, MA), anti-ICE1 antibodies (1:1000 dilution, HPA054452; Sigma-Aldrich Corp., St. Louis, MO), anti-ELL antibodies (1:1000 dilution, 14468; Cell Signaling Technology), anti-MED1 antibodies (1:1000 dilution, ab64965; Abcam, Cambridge, UK), anti-MED23 antibodies (1:1000 dilution, A300-425A; Bethyl Laboratories, Montgomery, TX), anti-Rpb1-NTD antibodies (1:2000 dilution, 14958; Cell Signaling Technology), anti- phospho-Rpb1-CTD (Ser5) antibodies (1:2000 dilution, 13523; Cell Signaling Technology), anti-phospho-Rpb1-CTD (Ser7) antibodies (1:2000 dilution, 13780; Cell Signaling Technology), anti-RNA polymerase II-CTD (phospho-2) antibodies (1:1000 dilution, ab5095; Abcam), anti-Coilin antibodies (1:2000 dilution, 14168; Cell Signaling Technology), anti-NPAT antibodies (1:300 dilution, sc-136007; Santa Cruz Biotechnology), anti-LSM11 antibodies (1:500 dilution, HPA039587; Sigma-Aldrich Corp.) and anti-β-actin antibodies (1:2000 dilution, sc-47778; Santa Cruz Biotechnology, Inc.).

Techniques: Mutagenesis, Microscopy, Staining, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Skap2 is required for β 2 integrin–mediated neutrophil recruitment and functions

doi: 10.1084/jem.20160647

Figure Lengend Snippet: The Skap2/WASp complex triggers actin polymerization, thereby enabling β 2 integrin activation by recruitment of talin-1 and kindlin-3. (A and B) F-actin polymerization of WT, Skap2 −/− , Was −/− , and Skap2 −/− Was −/− neutrophils (A) and Skap2 −/− neutrophils reconstituted with vector control, Skap2, or Skap2 W336K (B) after stimulation with CXCL1 in solution. n = 3. (C and D) Soluble ICAM-1 binding (C) and LFA-clustering (D) of CXCL1-stimulated WT, Skap2 −/− , and Was −/− neutrophils after pretreatment with DMSO or Lat. A. For clustering, 50 cells/experiment were analyzed. n = 3. (E) Knockdown of WASp in HL-60 cells. Quantification is shown on the right. n = 3. (F–I) Control, Skap2, or WASp knockdown HL60 cells were pretreated with DMSO or Lat. A and left unstimulated, plated on E-selectin with shear, or stimulated with IL-8 in solution. Lysates were incubated with GST fusion proteins of the β 2 integrin cytoplasmic domain (F and G) or immunoprecipitated with anti–β 2 integrin antibody (H and I). Precipitates were immunoblotted with anti–talin-1 and anti–kindlin-3 or anti–β 2 integrin antibody, respectively. Input was immunoblotted with anti-GAPDH antibody. Quantifications are shown below. n = 3. *, P < 0.001; # , P < 0.05 versus all other groups; one-way ANOVA (A–B and F–I), two-way ANOVA (C and D), or Student's t test (E). Data are means ± SEM. Ctrl, control; E-Sel., E-selectin; IP, immunoprecipitate; PD, precipitate.

Article Snippet: Incubation with anti-WASp (clone H-250; Santa Cruz Biotechnology, Inc.), Alexa Fluor 405–conjugated anti-WASp (clone B-9; Santa Cruz Biotechnology, Inc.), anti-Skap2 (Proteintech), or anti–talin-1 (clone 8d4) with corresponding isotype control antibodies was performed at 4°C overnight.

Techniques: Activation Assay, Plasmid Preparation, Binding Assay, Incubation, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Skap2 is required for β 2 integrin–mediated neutrophil recruitment and functions

doi: 10.1084/jem.20160647

Figure Lengend Snippet: Integrin activation is dependent on WASp-mediated de novo actin polymerization but also on PIP 2 synthesis. (A–C) F-actin polymerization (A), soluble ICAM-1 binding (B), and LFA-1 clustering (C) of Was −/− neutrophils reconstituted with WASp, vector control, or a WASp mutant lacking the VCA domain (WASpΔVCA) after stimulation with CXCL1 in solution. n = 3. (D and E) PIP 2 (D) and PIP 3 (E) levels of WT, Skap2 −/− , and Was −/− neutrophils after CXCL1 stimulation in solution or plating on E-selectin with shear. n = 2. (F) Subcellular localization of talin-1 in unstimulated or CXCL1-stimulated WT neutrophils pretreated with DMSO, PAO, or Lat. A. Representative images and statistics of talin-1 plasma membrane localization are shown. 40 cells/experiment were analyzed. n = 3. (G) Immunoprecipitation of β 2 integrin in IL-8– or E-selectin–stimulated HL-60 cells after pretreatment with DMSO or PAO. Precipitates were immunoblotted with anti–talin-1 and anti–kindlin-3 or anti–β 2 integrin antibody. Input was immunoblotted with anti-GAPDH antibody. Quantification is shown below. n = 3. *, P < 0.05; one-way ANOVA (A and D–F), two-way ANOVA (B and C), or Student's t test (G). Data are means ± SEM. Ctrl, control; E-sel., E-selectin; IP, immunoprecipitate; MSCV, murine stem cell virus.

Article Snippet: Incubation with anti-WASp (clone H-250; Santa Cruz Biotechnology, Inc.), Alexa Fluor 405–conjugated anti-WASp (clone B-9; Santa Cruz Biotechnology, Inc.), anti-Skap2 (Proteintech), or anti–talin-1 (clone 8d4) with corresponding isotype control antibodies was performed at 4°C overnight.

Techniques: Activation Assay, Binding Assay, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: Theranostics

Article Title: MGAT3-mediated glycosylation of tetraspanin CD82 at asparagine 157 suppresses ovarian cancer metastasis by inhibiting the integrin signaling pathway

doi: 10.7150/thno.43865

Figure Lengend Snippet: CD82 glycosylation at N157 promotes CD82 binding to integrin α5β1 and disrupts the integrin-fibronectin signaling pathway. A. Coupled immunoprecipitation (anti-Flag antibody; IP: Flag) and western blot analysis (anti-α5, β1 and β4 antibodies) of stable ES2 cells overexpressing Flag-tagged CD82, N157Q and N198Q mutants. B. Cell adhesion assay on fibronectin pre-coated plates that the stable control, CD82 or N157Q overexpressing cells were plated for 30 min. Adherent cells were visualized by Calcein fluorescence. A representative picture of three independent experiments is shown on the left. Scale bar 100 µm. Quantification of the relative fluorescence ± SD of three independent experiments is shown in the histogram on the right. ***p<0.005. NS: Not significant. C. Coupled immunoprecipitation (anti-β1 integrin antibody; IP: β1) and western blot analysis (anti-fibronectin; FN, α5, β1, FAK and Talin antibodies) of stable control (MCS) and CD82 overexpressing cells grown in presence of fibronectin. IgG was used as a negative control. A representative western blot out of three independent experiments is shown. D. Coupled immunoprecipitation (anti-α5 integrin antibody; IP: α5) and western blot analysis (β1, Paxillin, Talin, and anti-Flag antibodies) of cellular lysates derived from stable ES2 cells expressing CD82, N157Q and N198Q. A representative picture of three independent experiments is shown. E. Western blot analysis of phosphorylated and total FAK and Src in stable control and CD82 overexpressing cells at different time points after incubation with Fibronectin. A representative western blot of three independent experiments is shown. Note the reduction in the phosphorylation of FAK (p-FAK) and Src (p-Src) in CD82 as compared with MCS and N157Q expressing cells. F. Representative western blot (n=3) of phosphorylated and total FAK and phosphorylated paxillin (p-Paxillin) in stable ES2 cells upon treatment with fibronectin. Actin was used as a loading control. G. Representative immunofluorescence pictures of the subcellular localization of exogenous CD82 (Flag) and FAK in MCS, CD82 and N157Q overexpressing ES2 cells. Cellular nuclei were counterstained with DAPI. Scale bar 10 µm. H. Immunofluorescence analysis of Paxillin subcellular localization in MCS, CD82 and N157Q overexpressing cells (green). Exogenous CD82 was stained using anti-Flag antibody (red). Cell nuclei were counterstained with DAPI. Scale bar 10 µm. A representative picture of three independent experiments is shown.

Article Snippet: In these studies the following primary antibodies were used: Anti-Flag tag (Sigma-Aldrich; F1804), CD82 (CST; D7G6H), integrin β1 (SCBT; sc-8978), integrin α5 (CST; 4705S), p-FAK (CST; 3283S), FAK (CST; 3285S), p-SRC (CST; 2105S), SRC (CST; 2108S), paxillin (BD; 610620) integrin β4 (SCBT; sc-9090), Talin (SCBT; SC-15336), MGAT3 (proteintech; 17869-1-AP), Fibronectin (BD; 610078), Caveolin (SCBT; sc-984), Alix (SCBT; SC-99010), TSG101 (SCBT; SC-22774), p62 (SCBT; sc-166870), GM130 (SCBT; sc-55591).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Cell Adhesion Assay, Fluorescence, Negative Control, Derivative Assay, Expressing, Incubation, Immunofluorescence, Staining

Journal: Journal of Diabetes Research

Article Title: Protective Role of Tangshen Formula on the Progression of Renal Damage in db/db Mice by TRPC6/Talin1 Pathway in Podocytes

doi: 10.1155/2020/3634974

Figure Lengend Snippet: TSF protects podocyte-associated protein talin1 in db/db mice, which may involve increased expression of TRPC6 in podocytes. (a) Immunoblotting and quantitative analysis of talin1 ( n = 3 experiments). (b) Immunofluorescence costaining (scale bars, 25 μ m) of nephrin (red) and talin1 (green) showed that talin1 had a specific deletion in podocytes. (c) Immunofluorescence costaining (scale bars, 25 μ m) of nephrin (red; red arrow) and TRPC6 (green) showed that increased expression of podocyte TRPC6 in db/db mice (yellow arrow) compared to db/m mice and db/db + TSF mice ( n = 6). Fluorescent images were collected and assessed using a high-content screening system. The data were expressed as the mean ± SEM. # P < 0.05, ## P < 0.01 vs. db/m group; ∗ P < 0.05, ∗∗ P < 0.01 vs. db/db group. Statistically analyzed via a one-way ANOVA with Dunnett's correction.

Article Snippet: Talin1 antibody (14168-1-AP), NFATC2 (nuclear factor of activated T cell) antibody (22023-1-AP), NFATC3 antibody (18222-1-AP), integrin β 1 antibody (26918-1-AP), and GAPDH antibody (60004-1-Ig) were purchased from Proteintech (Wuhan, HB, China).

Techniques: Expressing, Western Blot, Immunofluorescence, High Content Screening

Journal: Journal of Diabetes Research

Article Title: Protective Role of Tangshen Formula on the Progression of Renal Damage in db/db Mice by TRPC6/Talin1 Pathway in Podocytes

doi: 10.1155/2020/3634974

Figure Lengend Snippet: TSF improved TRPC6-dependent Ca 2+ accumulation-mediated reduction of focal adhesions in AGEs-stimulated primary mice podocytes. (a) Effects of TSF or SAR7334 on intracellular Ca 2+ level of AGEs-induced primary mice podocyte injury ( n = 3 experiments). (b) Immunoblotting and quantitative analysis of talin1 ( n = 3 experiments). (c) Immunoblotting and quantitative analysis of TRPC6 and integrin β 1 ( n = 3 experiments). (d) Representative immunofluorescence stained with talin1 (green) and paxillin (red) (scale bars, 50 μ m). The data were expressed as the mean ± SEM of three independent experiments performed in triplicate. # P < 0.05, ## P < 0.01 vs. BSA group; ∗ P < 0.05, ∗∗ P < 0.01 vs. AGEs group; Δ P < 0.05, ΔΔ P < 0.01 vs. AGEs + TSF group. Statistically analyzed via a one-way ANOVA with Dunnett's correction.

Article Snippet: Talin1 antibody (14168-1-AP), NFATC2 (nuclear factor of activated T cell) antibody (22023-1-AP), NFATC3 antibody (18222-1-AP), integrin β 1 antibody (26918-1-AP), and GAPDH antibody (60004-1-Ig) were purchased from Proteintech (Wuhan, HB, China).

Techniques: Western Blot, Immunofluorescence, Staining