Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Schematic shows the morphology of sarcomere disassembly. B. Schematic of Co-IP/MS by TNNT2 pulldown. C. Table lists selected TNNT2 associated proteins identified from the Co-IP/MS. * PSM: Peptide Spectrum Matches. Number of spectra assigned to peptides that contributed to the inference of the protein. **MIC Sin: The normalized spectral index statistic for the protein for the specific group. Calculated from the intensity of fragment ions in each spectrum assigned to a protein. ***Ratio: Quantitative ratio for protein between groups derived from the MIC Sin value. D. Coimmunoprecipitation of ADD1 from total heart extracts at P1 and P7, probed for TNNT2 and α-actinin. E. (Left) Representative WB to show endogenous expression profile of ADD1 and ADD3 at different ages. (Right) Quantification of adducin expression level relative to internal control GAPDH. (n=3 per group). F. Expression of four selected cytoskeletal proteins from MS results, α-, γ-adducin, spectrin, and filamin were stained in neonatal hearts, showing profile of proliferation (left) and non-proliferation (right) cardiomyocytes. Arrows point to representative cells. Arrow heads in filamin staining images refer to nucleus location. Scale bar=10 μm.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Expressing, Control, Staining

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. (Top) Schematic of exons and introns of Add1 gene. Red rectangle represents exon 15 which is the target of alternative splicing. (Bottom) Schematic shows inclusion or exclusion of exon 15 in mRNA alternative splicing events. The corresponding translated protein schematic is shown next to the mRNA. Inclusion of exon 15 has an in frame stop code which generates ADD1 isoform 2, composing 636 amino acids (a.a.). Exclusion of exon 15 translates into ADD1 isoform 1, composing 732 a.a. B. Western blot shows endogenous profile of ADD1 isoform 2 at different ages. GAPDH is used as internal control. C. Representative images of NRVM transduced with AAV6-Add1i1 (i, ii), AAV6-Add1i2 (iii, iv), and AAV6-GFP (v) at day 3. Schematics of AAV expression cassettes are shown with each group images. Cells were fixed and immunostained with α-actinin (ACTN2, red) to reveal sarcomere organization. Green fluorescence represents either Adducin isoform 1 and 2, or GFP. High magnification images of squared regions are shown on the right. Dotted lines indicated the boarder of cardiomyocytes. (i) and (iii) represents partial disassembled sarcomeres. (ii) and (iv) represents fully disassembled sarcomeres. (v) represents assembled sarcomeres. Percentage of sarcomere assembly changes by AAV are shown in the graph. D. (Top) Schematic to show the strategy of generating Add1 isoform 2 single transgenic line. (Bottom left) H&E staining of WT and cardiac-specific Add1 i2 transgenic. Scale bar = 1 mm. (Bottom right) Heart weight to body weight ratio in control and cardiac-specific Add1i2 transgenic. E. (Top) Images to show expression pattern of ADD1 i2 transgenic at P14 and P28. (Bottom) High magnification images of squared region from the top panels. Scale bar = 10 μm. F. (Top) Representative images to show sarcomere pattern from ADD1 i2 transgenic at P14 and P28. Insets are high magnification images of single cardiomyocyte (squared regions). Dotted lines are drawn around outer and inner borders of sarcomeres. (Bottom) Quantification graph to show the extent of sarcomere disassembly. Disassembly scores were defined as: (Outer Area – Inner Area) / Outer Area. Age matched litter maters of WT mice were used as controls. At P14, ADD1 i2 transgenic mice have significantly thinner sarcomeric zone with central clearance compared to controls. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Alternative Splicing, Western Blot, Control, Transduction, Expressing, Fluorescence, Transgenic Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Endogenous phospho-adducin expression pattern in neonatal mouse heart. Schematics of phosphosite locations is shown on the left. Right: immunostaining images show co-staining images of anti-phospho-site antibody with sarcomeric protein TNNT2 or α-actinin. From top to bottom are Phospho-sites T445/T480, S12/S355, S714/S724, and S408/S436/S481. Arrows indicate phospho-adducin colocalized with disassembled sarcomeres. Arrow heads indicate colocalization with assembled sarcomeres. B. Endogenous expression of phospho-adducin T445 in NRVM as stained with α-actinin (red), anti-phospho-adducin pT445 (green) and DAPI. Phospho-adducin translocates from the nucleus to the cytoplasm during mitosis. C. (Top): WB examination of endogenous phospho-T445 expression level. (Bottom) Quantification of pT445 expression level in relative to ADD1 expression level. D. Overexpression of adducin phospho- or non-phospho mimic T445/T480 in NRVM as induced by AAV6. (Top left) Schematic diagram of AAV shuttle vector shows that adducin mutants are expressed with 3 tandem FLAG epitopes in frame at the N-terminus. (Bottom left) Immunofluorescent staining with α-actinin antibody to show sarcomeric structure. Adducin mutants are detected by Flag antibody in green. High magnification images of representative region (white box) were shown on the right. Dotted lines were drawn around the cell boarder. (Right) Quantitative analysis of disassembled sarcomeres induced by adducin overexpression. Scale Bar = 10 μm. E. (Top) Schematics to show strategy of generating cardiac specific inducible Add1 i1 phospho single transgenic (p-TRE T445E/T480E, α-MHC tTA). (Bottom left) H&E staining of control and cardiac-specific Add1 i1 phospho transgenic. Scale bar = 1 mm. (Bottom right) Heart weight to body weight ratio in control and cardiac-specific phospho-single transgenic. F. (Top) Images to show expression pattern of phospho-Add1i2 transgenic at P14 and P28. (Bottom) High magnification images of selective phospho-adducin mimic overexpression cardiomyocytes. At P14, we saw phospho-mimic expresses both in cytoplasm and on membrane (Bottom left). At P28, phospho-mimic adducin expresses on membrane and in nucleus (Bottom right). Scale bar = 10 μm. G. (Left) Representative images to show sarcomere pattern from phospho-ADD1 i1 transgenic at P14 and P28. Insets are high magnification images of single cardiomyocyte. Dotted lines are drawn around outer and inner edges of cardiomyocytes. (Right) Quantitative graph to show sarcomere disassembly. Disassembly scores were defined the same way as that of Add1i2 transgenic in . Scale bar = 10 μm. Scale bar= 10 μm. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Expressing, Phospho-proteomics, Immunostaining, Staining, Over Expression, Plasmid Preparation, Transgenic Assay, Control, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Phospho-adducin expression profile in regenerating neonatal heart at P4 by anti-pT445 staining. Note that the expression of pT445 translocates from nucleus to cytoplasm during cardiomyocyte mitosis. Scale bar= 10 μm. B & C. Overexpression of adducin mutants S12/S355 ( B ), and S714/724 ( C ) in NRVM induced by AAV6. (Top) Schematic diagram of AAV shuttle vectors for adducin expression. Mutants are expressed with 3 tandem FLAG epitopes in frame at the N-terminus. Middle. Immunofluorescent staining with α-actinin antibody (red) to show sarcomeric structure. Adducin mutants are detected by Flag antibody in green. Insets show high magnification images of boxed region. Bottom. Quantitative analysis of disassembled sarcomeres induced by adducin overexpression. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Expressing, Staining, Over Expression, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. HEK293T cells were transfection of variant 1(p-3xFlag-Add1i1) and Add1 variant 2 (p-3xFlag-Add1i2) in the presence or absence of Add3 (p-3xTy1-Add3). 24 hours after the transfection, the cells were treated with 80 μM cycloheximide (CHX) to inhibit protein translation. Cells were collected 0, 24, and 48 hours after treatment. (Left) WB to show protein level. (Right) Measurement of relative ADD1i1 or Add1i2 protein level (calculated as FLAG vs GAPDH) in the absence or presence of ADD3. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a one-tailed t -test. B. Schematic diagram to show the constructs of generating Add1 i2 and Add3 double transgenic. Both expression constructs were driven under an α-MHC promoter. C. QPCR results to show the mRNA level of Add1i2 in 2-month-old double transgenic with low expression. D. (Left) H&E staining of control and cardiac-specific Add1 i2/Add3 double transgenic with low expression. Samples are of two months old mice. Scale bar = 1 mm. (Right) Heart weight to body weight ratio in WT and low expression double transgenic. E. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in 2-month old ADD1 i2/ADD3 double transgenic (Low expression). n=3 for each group. F. Longitudinal views to show sarcomere pattern from 2 months old WT and Adducin double transgenic (Low expression). The high magnification images of boxed region in low magnification image are shown on the right. Dotted lines the border where adducin is expressed. Inset shows partial disassembled sarcomeres (disorganized but not absent sarcomeres). The corresponding adducin expression are indicated by arrows. Scale bar= 10 μm. G. QPCR results to show the mRNA level of Add1i2 in 2-month-old double transgenic with high expression. H. (Left) H&E staining of control and cardiac-specific Add1 i2/Add3 double transgenic with low expression. Samples are of 2-month-old mice. Scale bar = 1 mm. (Right) Heart weight to body weight ratio in WT and high expression double transgenic. I. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in 1-month-old ADD1 i2/ADD3 double transgenic (high expression). (n=3 for each group). J. (Top) Representative images to show sarcomere pattern from 1-months old ADD1 i2/ADD3 double transgenic (high expression). (Bottom) Enlarged images of Insets from above panels. K. (Left) ADD1 i2/ADD3 double transgenic with high expression has evidence of cardiomyocytes fragmentation. (Right) High magnification of boxed region on the left. Arrows point to sarcomere fragments where ADD1 and TNNT2 are colocalized. Scale bar = 10 μm. Scale bar= 10 μm. Data are presented as mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a two-tailed t -test.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Transfection, Variant Assay, One-tailed Test, Construct, Transgenic Assay, Expressing, Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. (Top) Schematic diagram of the constructs of generating phospho-mimic Add1 i2 and Add3 double transgenic. Glu substitution on T445 and T480 mimic phosphorylation form of Add1 i2. Both expression constructs were driven under an α-MHC promoter. (Bottom) H&E staining of control and cardiac-specific phospho-Add1 i2/Add3 double transgenic. Samples are of two-month-old mice. Scale bar = 1 mm. B. Heart weight to body weight ratio in WT and phospho double transgenic at P21 days and 2 months. (n=3 for P21, and n=5 for 2 months). C. Echocardiography (Left) and left ventricular systolic function quantification by ejection fraction (Right) in P21 and 2 months old ADD1 i2/ADD3 double transgenic (Low expression). (n=3 for P21, and n=5 for 2 months). D. QPCR of mRNA level of Add1i2 in 2-month-old phospho-double transgenic. E. Sarcomere pattern of phospho-double transgenic at P7 (i), P14 (ii), P21 (iii) and 2-month-old (iv). The high magnification images of boxed region are shown on the right side of zoom out image. Dotted lines circle out the boarder where adducin expresses outside sarcomeres. Arrows refer to regions with high level of adducin expression. Note the clearance of sarcomeres where adducin is expressed. Scale bar= 10 μm. F. Large scale kinase screening assay identified 7 candidates that potentially phosphoorylate T445 (left) and T480 (right) sites. Corrected kinase activity values (raw value minus sample peptide background, in cpm) with sample peptide at one concentration in 245 Ser/Thr peptide kinase assays. Blue: Kinase with sample peptide; yellow: Kinase activity w/o sample peptide. G. Serine/Threonine Kinases Hit Assay to verify 3 kinases, NEK1, DCAM1 and IRAK4, selected from initial kinase screening assay. Each kinase was tested at three concentrations in triplicates. These results suggested that the kinases NEK1, DCAMKL2, IRAK4 all show a concentration-dependent ability to phosphorylate the sample peptide “T445” and “T480”. H. Validation of endogenous expression pattern of IRAK4 (Left) and NEK1 (Right) in the regenerating neonatal heart. Positive signals are indicated by arrow. I. (Top) Schematic of IRAK4 expression cassettes in AAV6 constructs. Overexpress IRAK4 (green) in NRVM causes sarcomere (stained with α-actinin, red) disassembly (i) and pT445 adducin (stained with pT445, red) translocated from nucleus to cytoplasm (ii). Arrowheads indicate adducin is expressed in the nuclei in IRAK4 negative cells. Scale bar = 10 μm. J. (Top left) Schematic of IRAK4 expression cassettes in AAV9 constructs. (Top right) Schematic drawing of experimental design. (Bottom) AAV9-IRAK4-GFP was injected into CD1 pups at P1 and hearts were collected at P15 for sectioning and staining. IRAK4 overexpression caused strong sarcomere disassembly (i) in cardiomyocyte compared to littermate controls (ii).

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Construct, Transgenic Assay, Phospho-proteomics, Expressing, Staining, Control, Screening Assay, Activity Assay, Concentration Assay, Biomarker Discovery, Injection, Over Expression

Journal: bioRxiv

Article Title: Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

doi: 10.1101/2020.12.24.424022

Figure Lengend Snippet: A. Table lists selected ADD1 associated proteins identified from the Co-IP/MS. * PSM: Peptide Spectrum Matches. Number of spectra assigned to peptides that contributed to the inference of the protein. **MIC Sin: The normalized spectral index statistic for the protein for the specific group. Calculated from the intensity of fragment ions in each spectrum assigned to a particular protein. ***Ratio: Quantitative ratio for protein between groups derived from the MIC Sin value. Scale bar=10 μm. B . Schematic of the purification process of short and long adducin complexes. C. Schematic drawing shows the flowchart of Co-IP/MS by purified adducin complex pulldown D. Bar graph for Canonical Pathways enriched in each sample. X-axis shows transformed adjusted p value (Benjamini-Hochberg Procedure) of overlap between sample proteins and pathways. FDR refers to false discovery rate. *P<0.05, **P<0.01, ***P<0.001; Statistical significance was calculated using a two-tailed t -test. E. Network plot of cytoskeleton proteins from the pull down assay interact with adducin_long or adducin_short at different time points (P1 and P21). Different color labeling highlighting common and unique proteins among sample groups. Red color represents the four sample groups in the pulldown assay. Yellow color represents proteins common to all groups. Green represents proteins common to only two or three groups. Blue represents proteins unique to each group. F. Proximity ligation assay (PLA) to examine direct interaction between adducin (pT445) and sarcomeric proteins (α-actinin 2) in P4 heart tissue. The red dots (arrows) are fluorescent signals which indicate a close interaction between two antigens. Samples include Interaction between α-actinin and troponin T (as positive control) or phospho-adducin (pT445) and α-actinin. Scale bar=10 μm. G. Immuno-EM image to show the subcellular location of phospho-adducin in cardiomyocytes with disassembled sarcomeres in neonatal MI hearts at day 3 after surgery. Two red boxes in left image are magnified on the right. (i) adducin is localized to z-disks. (ii) phospho-adducin is heavily localized to z-disks associated with plasma membrane in cardiomyocytes with disassembled sarcomeres. Red arrows indicate location of adducin.

Article Snippet: Antibodies used in WB are anti-α-adducin antibody (Santa Cruz H-100, sc-25731, 1:500), anti-γ-adducin antibody (Santa Cruz H-60, sc-25733, 1:500), anti-FLAG antibody (Genescript #A00187, 1:3000), anti-Ty1 antibody (Diagenode, #C15200054, 1:3000) and Peroxidase AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch 711-035-152, 1:20,000), Peroxidase AffiniPure goat antimouse IgG (Jackson ImmunoResearch 115-035-003, 1:20,000) were processed by LI-COR.

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Purification, Transformation Assay, Two Tailed Test, Pull Down Assay, Labeling, Proximity Ligation Assay, Positive Control, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

doi: 10.1101/2022.11.29.518315

Figure Lengend Snippet: (a) Immunoblotting of cleaved caspase 7 following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNα and 1000 U/mL TNFα) (Mix) for 96 h. Tubulin was used as loading control (n=3). (b) Immunoblotting of IκBα, phosphorylated STAT1, and phosphorylated JNK following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNγ and 1000 U/mL TNFα) alone or in combination (Mix) for 30 min. GAPDH was used as loading control. Representative image (n=3). Lysates from INS-1E cells with/without cytokine exposure were used as positive control. Gene expression of (c) DDIT3/CHOP, (d) HRK/DP5 and (e) CXCL10 in cells exposed to individual cytokines for 48 h. GAPDH was used as housekeeping gene. Data are means ± SEM (n=4), *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-MHC-I (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-GAPDH (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076) or anti-rabbit (#7074) (Cell Signaling) IgG antibodies.

Techniques: Western Blot, Control, Positive Control, Gene Expression