Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. qPCR results from pig articular chondrocytes stimulated with OSM and treated with or without SRC inhibitor (SU6656) for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein expression of pSRC in ATDC5 cells after transfection (72 hours) with the modified and WT gp130 plasmids stimulated with OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. qPCR results from ATDC5 cells treated with SRC inhibitor (SU6656) alone, or transfected with indicated variants of plasmids and stimulated with or without OSM for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. D. Protein expression of gp130 pY814 in untreated fetal (17 weeks) and adult articular chondrocytes. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Transfection, Modification

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of pSRC in pig articular chondrocytes stimulated with IL-6 cytokines. Horizontal lines with bars show the mean ± SD. n=3. B. Schematic of modified amino acids within gp130 812-827 domain. Protein expression of pSRC in Ba/F3 cells after transfection with the modified or WT gp130 plasmids stimulated with IL-6 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. CRISPR gRNA was designed by PNA Bio. Y814 mutant mouse (F814) was generated by the USC transgenic mouse core. Mice genotyping was performed by GeneWiz. Representative images of 4-month-old males shown. n=4. D. Protein expression of gp130 pY814 in wild type (WT) and F814 mouse splenocytes treated with and without OSM and LIF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. E. Distribution of differentially expressed genes in MA plot of wild type (WT) and F814 mouse periarticular stromal cells. MA plots (log2fold change vs log2mean expression) for WT-OSM vs WT and Y814-OSM vs Y814 mouse periarticular stromal cells. Each dot represents a gene. Red dots represent upregulated genes i.e. log2fold-change >1 and p-value<0.05. Blue dots represent downregulated genes i.e. log2fold change < −1 and p-value 0.05. Genes that do not qualify this threshold are indicated by grey dots. F. Levels of protein complex formation between gp130 and pSRC in wild type and F814 mouse splenocytes stimulated with or without OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. G. Protein expression of pSRC in wild type and F814 mouse splenocytes stimulated with or without OSM for 24 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Modification, Transfection, CRISPR, Mutagenesis, Generated, Transgenic Assay

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Protein expression of gp130 pY814 in wild type (WT) and F814 mouse splenocytes treated with and without OSM and LIF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein levels of pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. C. Computational analysis using GOLD software predicted a high affinity binding of peptide QQpYF to the regulatory site of SRC (c-SRC). c-SRC as visualized by crystallography. The structure of the indicated c-SRC domains is shown in ribbon diagram representation (left) as well as with electrostatic potential (blue, positive charge; red, negative charge; white, neutral) mapped onto the molecular surface (right). Peptide QQpYF is shown in stick representation. D. Transcription of genes was determined via qPCR in human adult OA articular chondrocytes treated with or without peptide QQpYF for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. E. Pig knee cartilage explants were stimulated with or without OSM and treated with or without peptide QQpYF at indicated doses for 72 hours followed by a neoepitope assay. Levels of cleaved ACAN and COL2 neoepitopes in the supernatant were quantified with respect to the wet weight of the explant. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Software, Binding Assay

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Protein levels of pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of control scrambled peptide or peptide QQpYF for 4 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques:

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of gp130 pY814 in pig articular chondrocytes treated with or without OSM and R805 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3. B. Gross morphology and Masson’s trichrome staining of bearded dragon treated with vehicle control and R805 14 days post-amputation. Dashed lines mark amputation planes. n=10. sc=spinal cord; we=wound epithelium; ve=vertebra. C. Gross morphology of bearded dragon co-treated with vehicle control or R805 and PBS or clodronate liposomes 14 days post-amputation. Dashed lines mark amputation planes. n=10. D. Mouse vehicle-treated or R805-treated post-wound day (PWD) 21 wound sections. Representative images are shown. n=8. E. Hair follicle (n=3) and fiber length (n=8) in R805-treated mouse PWD 14 and untreated control wounds. Horizontal lines with bars show the mean ± SD. F. Re-clustering of clusters annotated as macrophages from skin wounds of vehicle-treated or R805-treated mice PWD 14. Dot plots depict gene expression in each macrophage cluster. The contribution of each sample to each cluster is shown as a stacked bar graph. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. DCs = dendritic cells. G. Re-clustering of clusters annotated as fibroblasts from skin wounds of vehicle-treated or R805-treated mice PWD 14. Dot plots depict gene expression in each fibroblast cluster. The contribution of each sample to each cluster is shown as a stacked bar graph.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Receptor-competition assay. Adult human articular chondrocytes were treated with or without indicated IL-6 family of cytokines and different doses of R805 for 4 hours. Co-IP measuring protein expression was performed after transfection with gp130 FLAG plasmid (72 hours) followed by western blot with respective receptor antibodies. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Competitive Binding Assay, Co-Immunoprecipitation Assay, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Receptor-competition assay. Adult human articular chondrocytes were treated with or without indicated IL-6 family of cytokines and different doses of R805 for 24 hours. Co-IP measuring protein expression was performed after transfection with gp130 FLAG plasmid (72 hours) followed by western blot with respective receptor antibodies. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Competitive Binding Assay, Co-Immunoprecipitation Assay, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: A. Protein expression of gp130 pY814 in pig articular chondrocytes treated with or without OSM and R805 for 4 hours. Horizontal lines with bars show the mean ± SD. n=3. B. qPCR results from pig articular chondrocytes cells treated with OSM with or without R805 for 48 hours. Horizontal lines with bars show the mean ± SD. n=3. C. Pig cartilage knee explants were incubated with or without OSM for 72 hours with or without R805 at indicated concentrations followed by a neoepitope assay. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: Signaling modality within gp130 receptor enhances tissue regeneration

doi: 10.1101/2022.01.05.475124

Figure Lengend Snippet: Levels of complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM in presence or absence of R805 for 24 hours. Horizontal lines with bars show the mean ± SD. n=3.

Article Snippet: The immune complexes were sedimented, washed and separated by SDS-PAGE (see below) and further analyzed by Western blot using p-SRC (pY416-SRC) (cat# 1246F, Novus Biologicals) or NEMO (cat # 18474-1-AP, Proteintech) antibodies and normalized to gp130 (cat #bs-1459R, Bioss) or total SRC (cat # 2123, Cell Signaling).

Techniques:

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 2. The expression of selected receptors (CXCR4, Leptin R, EGFR, IL2R gamma, and gp130) on cell membrane of selected breast cancer cell lines. gp130 was the only receptor that is overexpressed in selected triple negative breast cancer (TNBC) cell lines, MDA 231wild type (WT), MDA 231 K, and MDA 468 cell lines. The expression of the receptors was detected using fluorescent-labeled monoclonal antibodies to the selected receptors and flow cytometry. Asterisks indicates significant difference to non-TNBC cell lines (one-way ANOVA, Tukey post-hoc test; p<0.05). Bar graph represents the mean values (n = 4), and the error bars show the standard deviation.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Membrane, Labeling, Bioprocessing, Flow Cytometry, Standard Deviation

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 4. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected antiapoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1). The mRNA expression was determined using RT-PCR in MDA 231 (A) and MDA 468 cells (B). In both cell lines, the drop in mRNA level of all three antiapoptotic proteins was significantly higher in cells exposed to JAK2/gp130 siRNA combination. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. The effect of single and combinational silencing of gp130 and JAK2 on Bcl2 and Mcl-1 at protein level was confirmed by western blotting (C) in MDA 231 cells. β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of antiapoptotic related molecules was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 5. The effect of Individual and combinatorial silencing of selected proteins in JAK/STAT pathway on expression of selected pro-apoptotic proteins (PTEN and p53). The mRNA expression of the pro-apoptotic proteins was determined using RT-PCR in MDA-MB-231 (A) and MDA-MB-468 cells (B). The mRNA levels of both PTEN and P53 proteins showed the most significant increase in both cell lines after simultaneous silencing of JAK2/gp 130. Bar graphs represent the mean values (n = 3), and the error bars show the standard deviation. Th effect of single and silencing of gp130 and JAK2 on PTEN and P53 at protein level was confirmed by western blotting in MDA 231 cells (C). β-actin served as endogenous protein. A similar trend was noted at the protein level (D). Expression of pro-apoptosis proteins was normalized to untreated cells (NT). Bar graph represents the mean values (n = 3), and the error bars show the standard deviation. Asterisks indicates significant difference compared to all other study groups (one-way ANOVA, Tukey post-hoc test; p < 0.05).

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Combinational silencing of components involved in JAK/STAT signaling pathway.

doi: 10.1016/j.ejps.2022.106233

Figure Lengend Snippet: Fig. 9. The effect of silencing STAT3 and the individual and combinational silencing of JAK2 and gp 130 on STAT3 activation in MDA 231 cell line using differential Western Blotting on cytoplasmic and nuclear compartments. GAPDH and Histone H3 levels were analyzed as cytoplasmic and nuclear markers, respectively, which indicated effective isolation of the sub-cellular compartments. The bands representing STAT3 show total STAT3 levels (phosphorylated and non-phosphorylated). (A) Western blot showing the cytoplasmic expression level of STAT3 and p-STAT3. The expression of p-STAT3 was negligible in cytoplasm compared to the total STAT3 levels. (B) Bar graph represents the cytoplasmic expression level of STAT3 in different study groups (n = 3; error bars represent standard deviation). The STAT3 levels in cytoplasm dropped most significantly as a response to silencing STAT3. (C) Differential western blot shows the nuclear expression level of STAT3 and p-STAT3. The effect of silencing is apparent on both total STAT3 and the p-STAT3 levels. (D) Bar graph shows nuclear protein levels of p- STAT3 in different study groups (n = 3; error bars represent standard deviation). The drop in level of p-STAT in nucleus was most significant in cells exposed to JAK2/gp130 siRNA combination.

Article Snippet: Fluorescein antibodies for flow cytometry included CXCR4 (Catalog No FAB170F), Leptin R (Catalog No FAB867F), Epithelial growth factor receptor (EGFR; Catalog No FAB10951F), gp130 (Catalog No FAB228F), and IL2R (Catalog No FAB2842F) were purchased from R&D systems.

Techniques: Activation Assay, Western Blot, Isolation, Expressing, Standard Deviation

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: NS1 SC09 interacts with LRPPRC. (A) 293T cells were transfected with NS1 SC09 -Flag, NS1 JL89 -Flag, or empty vector. Cell lysates were pulled down using the antibodies against Flag tag. Proteins were then separated using SDS-PAGE and stained with silver, after which they were identified using LC/MS. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) HeLa cells were co-transfected with LRPPRC-Flag and NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Twenty-four h later, cells were assessed for the localization of NS1, LRPPRC, and TOMM20. Cyan color indicated the colocalization of LRPPRC and TOMM20 and white color indicated the colocalization of LRPPRC, NS1, and TOMM20. Scale bar: 5 μm. It was representative of 20 cells. (D) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, FLAG-tag, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: Summary annotated proteins identified to interact with NS1 SC09 -flag but not NS1JL89-flag and Vec-flag.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Virus, Binding Assay

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The key domain responsible for the interaction between NS1 SC09 and LRPPRC. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to HA-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: LRPPRC is a key factor that regulates the replication of IAVs through the autophagy pathway and is antagonized by NS1 SC09 . (A and B) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 0.01. The supernatants were sampled at 0, 12, 24, and 48 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates at 0, 12, 24, and 48 h post-infection were analyzed using western blotting. (C and D) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were infected with WSN at the MOI of 5.0, in the presence or absence of rapamycin (20 μm) or 3-MA (5 mM). The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. The cell lysates were analyzed using western blotting. (E) 293T cells, 293T_LRPPRC cells, and LPRRPC KO cells were transfected with mRFP-GFP-LC3B and then infected with WSN at the MOI of 0.1. Cells were analyzed to assess the formation of autophagosomes. Fluorescence signals indicated the expression of RFP and GFP (yellow color: incomplete autophagy, red color: complete autophagy). Scale bar: 10 μm. It was representative of 20 cells. (F) The graph shows the quantification of mRFP+GFP+ autophagosomes and mRFP+GFP- autolysosomes by taking the average number of dots in 20 cells (n=average number of dots in 20 cells). (G and H) 293T cells and LPRRPC KO cells were infected with WSN-NS1 SC09 or WSN at the MOI of 0.1. The supernatants were sampled at 24 h post-infection, and the virus titers were determined using endpoint titration in MDCK cells. Cell lysates were analyzed using western blotting. (I) 293T cells and LPRRPC KO cells were transfected with NS1 SC09 -HA or NS1 WSN -HA. 24 h post-transfection, cell lysates were analyzed using western blotting. Error bars indicate the SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection, Virus, Titration, Western Blot, Transfection, Fluorescence, Expressing

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The interaction between NS1 SC09 and LRPPRC results in initiation of BECN1-dependent autophagy. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (D) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-BECN1 antibody or control IgG. (E) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP. (F) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-PIK3C3 antibody or control IgG. (G) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: A proposed model for the pathway of autophagy induced by NS1 pdm09 . In the normal physiological state, LRPPRC interacts with the BECN1-BCL2 heterodimer, forming a stable complex in the mitochondria, thereby inhibiting BECN1-dependent autophagy. During A/(H1N1) pdm09 infection, the NS1 pdm09 promotes viral replication by enhancing autophagy which is mainly attributed to the inhibitory effect of NS1 pdm09 on the negative regulation of autophagy by LRPPRC. The interaction between NS1 pdm09 and LRPPRC competitively blocks the interaction of LRPPRC with BECN1-BCL2 heterodimer, resulting in degradation of BCL2 and increased recruitment of BECN1 by PIK3C3, and then induction of the initiation of autophagy.

Article Snippet: Antibodies used in this study include: mouse anti-HA tag monoclonal antibody (Sigma-Aldrich, H9658), mouse anti-Flag tag monoclonal antibody (Sigma-Aldrich, F1804), rabbit anti-HA tag polyclonal antibody (Sigma-Aldrich, F7425), mouse anti-ACTB monoclonal antibody (Sigma-Aldrich, A5441), rabbit anti-ACTB polyclonal antibody (ABclonal Technology, AC026), rabbit anti-LC3B monoclonal antibody (ABclonal Technology, A19665), rabbit anti-LAMP2 polyclonal antibody (ABclonal Technology, A0593) rabbit anti-BECN1 polyclonal antibody (Proteintech, 11,306-1-AP), rabbit anti-LRPPRC polyclonal antibody (Proteintech, 21,175-1-AP), rabbit anti-BCL2 monoclonal antibody (ABclonal Technology, A19693), rabbit anti-PIK3C3/VPS34 monoclonal antibody (ABclonal Technology, A12295), rabbit anti-NS polyclonal antibody (CUSABIO, H0228A), and NP monoclonal antibody (made by our lab).

Techniques: Infection

Journal: The International journal of developmental biology

Article Title: Activated Notch1 is a stronger astrocytic stimulus than leukemia inhibitory factor for rat neural stem cells.

doi: 10.1387/ijdb.092869nr

Figure Lengend Snippet: Fig. 1. Cortical NSC respond to LIF and CNTF stimulation. Proliferating neural progenitors/NSC expressed Nestin (A,B) together with GP130 (A) or LIFRβ (B). In control cultures, no signal for phospho (p)STAT3 was detected (C). After addition of LIF, NSC show nuclear pSTAT3. Accordingly, in immunoblot assays, control (CTL) cells have non-phosphorylated STAT3, and 10 ng/ml CNTF or the indicated LIF concentrations induced phosphorylation of STAT3 (D). The transcriptional activation of Socs3 is only found in cells stimulated with LIF or CNTF (E). Images are representative of 3 independent experiments performed in duplicate. Scale bars represent 20 μm for A and 50 μm for B. -RT, RNA without reverse transcriptase.

Article Snippet: Other cell markers were immunodetected with the following antibodies: Nestin (Developmental Studies Hybridoma Bank), β-Tubulin-III (mouse or rabbit TUJ1, Covance), GFAP (rabbit, DAKO; rat, Zymed), pSTAT3 (Cell Signaling), Sox2 (Chemicon), GP130 (Santa Cruz Biotechnology) and LIFRβ (Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Phospho-proteomics, Activation Assay, Reverse Transcription

Journal: The Journal of Biological Chemistry

Article Title: The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway

doi: 10.1074/jbc.RA120.013815

Figure Lengend Snippet: ZDHHC5/8 are critical for retrograde signaling by the Gp130/JAK/STAT3 pathway, but not the DLK/JNK pathway. A, DRG neurons cultured in microfluidic chambers were infected with the indicated viruses. Cultures were left untreated, or distal axons were axotomized by aspiration. Cell body chambers were then immunostained to detect phospho-c-Jun (p-c-Jun) and DAPI. B, quantified data from A reveal that axotomy-induced c-Jun phosphorylation, which is strongly DLK-dependent (28), is not affected by Zdhhc5/8 knockdown. ns, not significantly different from control-infected axotomized condition, two-way ANOVA: virus p = 0.2754 (F(1, 18) = 1.265), axotomy p < 0.0001 (F(1, 18 = 27.89), interaction p = 0.3892 (F(1, 18) = 0.7785), n = 5–6 determinations/condition. C, distal axonal chambers of DRG neurons cultured as in A were treated with or without 10 ng/ml rat CNTF, and cell body chambers were fixed and immunostained to detect STAT3 phosphorylation (pSTAT3) and DAPI. D, quantified data from C reveal that Zdhhc5/8 knockdown significantly reduces axotomy-induced STAT3 phosphorylation. n = 4 determinations/condition. **, p < 0.01, two-way ANOVA: virus p = 0.0119 (F(1, 12) = 8.766), treatment p = 0.0003 (F (1, 12) = 25.27), interaction p = 0.0166 (F(1, 12) = 7.74). E, Western blots of lysates from conventionally cultured DRG neurons (sister cultures of those used for microfluidic experiments in A–D) infected with the indicated viruses confirm effective reduction of ZDHHC5 and ZDHHC8 protein levels by their respective shRNAs (see also quantified data in Fig. S2). Bars, mean ± S.D. (error bars).

Article Snippet: Antibodies The following antibodies, from the indicated sources, were used: anti-ZDHHC5 (rabbit) (Sigma Prestige); anti-HA11 (16B12, mouse) (Covance); anti-GFP (clone 3E6, mouse) (Life Technologies); anti-ZDHHC8 (rabbit) (Santa Cruz Biotechnology, Inc.); anti-GM130 (rabbit) (Abcam); anti-Gap43 (rabbit) (Novus Biologicals); anti-pan-neurofascin (mouse) (NeuromAb); and anti-phospho-c-Jun (Ser-63, rabbit), anti-ERK1/2 (mouse), anti-GP130 (rabbit), and anti-phospho-STAT3 (Tyr-705, rabbit) (all from Cell Signaling Technologies).

Techniques: Cell Culture, Infection, Phospho-proteomics, Knockdown, Control, Virus, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway

doi: 10.1074/jbc.RA120.013815

Figure Lengend Snippet: Gp130 is palmitoylated in a ZDHHC5/8-dependent manner. A, HEK293T cells were transfected with an empty vector or with Gp130-expressing vector and subsequently treated with palmitoylation inhibitor 2-bromopalmitate (2BP) or vehicle. ABE fractions were prepared and blotted to detect palmitoyl-Gp130 (top). Lysates were blotted to detect total Gp130 expression (bottom). The image in the top panel is from a single Western blot analysis, cropped to remove intervening spacer lanes. The experiment shown is representative of three individual determinations. B, HEK293T cells were transfected with Gp130 cDNA plus the indicated ZDHHC5 or ZDHHS5 cDNAs and ABE assays performed. ABE fractions were blotted to detect palmitoyl-Gp130 (top). Lysates were blotted to detect total Gp130 and total ZDHHC5 expression (middle and bottom panels). C, quantified data for palmitoylated-to-total (Palm/total) Gp130 from B confirm that ZDHHC5 significantly increases Gp130 palmitoylation, whereas catalytically inactive ZDHHS5 does not. *, p < 0.05, nonparametric one-way ANOVA with Dunn's multiple-comparison post hoc test (p = 0.0086, Kruskal–Wallis statistic = 7.848), 4–5 determinations/condition. D, as in B, except that cells were transfected with the indicated ZDHHC8 or ZDHHS8 cDNAs, and lysates were blotted with Gp130 and ZDHHC8 antibodies. E, quantified data from D confirm that ZDHHC8 significantly increases palmitoyl-Gp130 levels, whereas catalytically inactive ZDHHS8 does not. *, p < 0.05, nonparametric one-way ANOVA with Dunn's multiple-comparison post hoc test (p = 0.0086, Kruskal–Wallis statistic = 7.848), 4–5 determinations per condition. F, cultured sensory neurons were infected with control virus or with viruses expressing ZDHHC5 and ZDHHC8 shRNAs. ABE fractions were prepared from lysates and blotted to detect palmitoyl-Gp130 and -GAP-43 (left panels). Lysates were blotted to detect total ZDHHC5, ZDHHC8, Gp130, GAP-43, and tubulin expression (right panels). G, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown significantly reduces Gp130 palmitoylation. *, p < 0.05, t test. H, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown does not significantly reduce Gp130 total levels. n.s., not significant, t test. I, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown does not significantly reduce Gp130 palmitoylation, when normalized to total Gp130. n.s., not significant, t test. Bars, mean ± S.D. (error bars).

Article Snippet: Antibodies The following antibodies, from the indicated sources, were used: anti-ZDHHC5 (rabbit) (Sigma Prestige); anti-HA11 (16B12, mouse) (Covance); anti-GFP (clone 3E6, mouse) (Life Technologies); anti-ZDHHC8 (rabbit) (Santa Cruz Biotechnology, Inc.); anti-GM130 (rabbit) (Abcam); anti-Gap43 (rabbit) (Novus Biologicals); anti-pan-neurofascin (mouse) (NeuromAb); and anti-phospho-c-Jun (Ser-63, rabbit), anti-ERK1/2 (mouse), anti-GP130 (rabbit), and anti-phospho-STAT3 (Tyr-705, rabbit) (all from Cell Signaling Technologies).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Comparison, Cell Culture, Infection, Control, Virus, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway

doi: 10.1074/jbc.RA120.013815

Figure Lengend Snippet: ZDHHC5/8 control Gp130 surface expression. A, surface fractions (left column) and total lysates from cultured DRG neurons infected with the indicated viruses were immunoblotted with the indicated antibodies. B, quantified data from A reveal that Zdhhc5/8 knockdown significantly reduces surface/total Gp130 (left histogram; *, p < 0.05, t test) but does not affect surface/total levels of the axonal palmitoyl-protein neurofascin (right histogram; n.s., nonsignificant, t test, n = 8 and n = 4 determinations/condition, respectively). Bars, mean ± S.D. (error bars).

Article Snippet: Antibodies The following antibodies, from the indicated sources, were used: anti-ZDHHC5 (rabbit) (Sigma Prestige); anti-HA11 (16B12, mouse) (Covance); anti-GFP (clone 3E6, mouse) (Life Technologies); anti-ZDHHC8 (rabbit) (Santa Cruz Biotechnology, Inc.); anti-GM130 (rabbit) (Abcam); anti-Gap43 (rabbit) (Novus Biologicals); anti-pan-neurofascin (mouse) (NeuromAb); and anti-phospho-c-Jun (Ser-63, rabbit), anti-ERK1/2 (mouse), anti-GP130 (rabbit), and anti-phospho-STAT3 (Tyr-705, rabbit) (all from Cell Signaling Technologies).

Techniques: Control, Expressing, Cell Culture, Infection, Knockdown

Journal: Neuro-Oncology

Article Title: Akt signaling pathway: a target for radiosensitizing human malignant glioma

doi: 10.1093/neuonc/nop059

Figure Lengend Snippet: Impact of STAT3 pathway down-modulation on SF763 cells. (A and D) Cells were treated with JSI-124 (7 hours) or with anti-gp130-blocking antibody (24 hours). Total proteins were electrophoresed by SDS–PAGE, followed by immunoblotting with anti-STAT3, anti-pSTAT3-Tyr705, and anti-β-actin antibody. (B) Clonogenic survival of SF763 cell line exposed 24 hours to a concentration range of JSI-124. One representative experiment performed in triplicate is shown. (C) Clonogenic survival of SF763 cell line exposed to 0.01 µM of JSI-124 during 24 hours. After 7 hours of treatment, cells were irradiated to 4 Gy and the surviving fraction was compared with that of control (DMSO). One representative of 3 independent experiments (performed in triplicate) is shown. (E) Clonogenic survival of SF763 cell line exposed to blocking anti-gp130 or to control (IgG2a) antibody when cells were attached in flasks (8 hours after seeding), before or after irradiation. Cells were irradiated to 4 Gy and the surviving fraction was compared with that of control (IgG2a). Representative experiment performed in triplicate is shown.

Article Snippet: All culture reagents were purchased from GIBCO (Invitrogen). gp130-blocking antibody (no. 852.060.000) and control (IgG2a, no. 857.080.000) mouse antibody were from Diaclone.

Techniques: Blocking Assay, SDS Page, Western Blot, Concentration Assay, Irradiation, Control

Journal: Neuro-Oncology

Article Title: Akt signaling pathway: a target for radiosensitizing human malignant glioma

doi: 10.1093/neuonc/nop059

Figure Lengend Snippet: Impact of STAT3 pathway down-modulation on SNB19 cells. (A) Cells were treated with anti-gp130-blocking antibody (24 hours). Total proteins were electrophoresed by SDS–PAGE, followed by immunoblotting with anti-STAT3, anti-pSTAT3-Tyr705, and anti-β-actin antibody. (B) Clonogenic survival of SNB19 cell line exposed to blocking anti-gp130 or to control (IgG2a) antibody when cells were attached in flasks (8 hours after seeding). Cells were irradiated to 2 Gy and the surviving fraction was compared with that of control (IgG2a). Representative experiment performed in triplicate is shown.

Article Snippet: All culture reagents were purchased from GIBCO (Invitrogen). gp130-blocking antibody (no. 852.060.000) and control (IgG2a, no. 857.080.000) mouse antibody were from Diaclone.

Techniques: Blocking Assay, SDS Page, Western Blot, Control, Irradiation

Journal: Stroke

Article Title: Neuroprotection by Interleukin-6 Is Mediated by Signal Transducer and Activator of Transcription 3 and Antioxidative Signaling in Ischemic Stroke

doi: 10.1161/strokeaha.111.626648

Figure Lengend Snippet: Figure 1. Increase in p-STAT3 (Y705) and Mn-SOD by IL-6 in mouse cerebral ischemic reperfusion in vivo and in vitro and disruption of IL-6R after ischemic reperfusion. Western blot analysis of p-STAT3 (Y705), Mn-SOD, and -tubulin after 3 hours of reperfusion after tFCI with or without IL-6 injections (A) or after 4 hours of reoxygenation after OGD with or without IL-6 treatments in cerebral cortical neurons (B). Summary graphs depicting the band intensity of each Western blot. *P0.05 versus sham (A) or control (B), #P0.05 ver- sus vehicle (n4 per group). Co-IP assays for analysis of association between IL-6R and gp130 (C) or between gp130 and p-STAT3 (D) after 3 hours of reperfusion after tFCI with or without IL-6 injections. Summary graphs depicting the band intensity of all Co-IP data. *P0.05 versus sham, #P0.05 versus vehicle (n4 per group). S indicates sham; IR, ischemic reperfusion; IP, immunoprecipitation; WB, Western blot; V, vehicle; C, control; OGD, oxygen–glucose deprivation/reoxygenation; OD, optical density; STAT3, signal trans- ducer and activator of transcription 3; Mn-SOD, manganese–superoxide dismutase; IL-6, interleukin-6; tFCI, transient focal cerebral is- chemia; IL-6R, IL-6 receptor.

Article Snippet: The primary STROKE/2011/626648 Supplemental Data / 3 antibodies we used were rabbit anti-IL-6R (1:100; Santa Cruz Biotechnology) and mouse anti- gp130 (1:100; R&D Systems).

Techniques: In Vivo, In Vitro, Disruption, Western Blot, Control, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Stroke

Article Title: Neuroprotection by Interleukin-6 Is Mediated by Signal Transducer and Activator of Transcription 3 and Antioxidative Signaling in Ischemic Stroke

doi: 10.1161/strokeaha.111.626648

Figure Lengend Snippet: Figure 3. Reduction of p-STAT3 and Mn-SOD by IL-6R knockdown in mouse cerebral cortical neurons. A, Western blot analysis of IL-6R, gp130, p-STAT3 (Y705), Mn-SOD, 3NT, and -tubulin in cerebral cortical neurons transfected with IL-6R- or gp130-specific siRNA or treated with AG490 for 24 hours. B, Summary graphs depicting the band intensity of each Western blot. *P0.05 (n4 per group). 3NT indicates 3-nitrotyrosine; OD, optical density; p-STAT3, phosphorylated signal transducer and activator of transcription 3; Mn-SOD, manganese–superoxide dismutase; IL-6R, interleukin-6 receptor; siRNA, small interfering RNA.

Article Snippet: The primary STROKE/2011/626648 Supplemental Data / 3 antibodies we used were rabbit anti-IL-6R (1:100; Santa Cruz Biotechnology) and mouse anti- gp130 (1:100; R&D Systems).

Techniques: Knockdown, Western Blot, Transfection, Small Interfering RNA

Journal: Stroke

Article Title: Neuroprotection by Interleukin-6 Is Mediated by Signal Transducer and Activator of Transcription 3 and Antioxidative Signaling in Ischemic Stroke

doi: 10.1161/strokeaha.111.626648

Figure Lengend Snippet: Figure 4. IL-6R upregulates transcriptional activity of Mn-SOD through STAT3 recruitment into the Mn-SOD promoter. ChIP assay for analysis of STAT3 recruitment into the mouse Mn-SOD promoter after 3 hours of reperfusion with or without IL-6 injection in the cerebral ischemic cortex (A) or in cerebral cortical neurons transfected with IL-6R- or gp130-specific siRNA for 24 hours (B). C, Luciferase assay for analysis of tran- scriptional activity of the Mn-SOD promoter in cerebral cortical neurons transfected with pGLu-Mn-SOD and IL-6R- or gp130- specific siRNA or treated with AG490 or IL-6 for 24 hours (n4 per group). Data are meanSEM *P0.05, **P0.001 versus control. IR indicates ischemic reperfusion; S, sham; V, vehicle; IL-6R, interleukin-6 receptor; Mn-SOD, manganese–superoxide dismutase; STAT3, signal transducer and activator of transcrip- tion 3; ChIP, chromatin immunoprecipitation; IL-6, interleukin-6; siRNA, small interfering RNA.

Article Snippet: The primary STROKE/2011/626648 Supplemental Data / 3 antibodies we used were rabbit anti-IL-6R (1:100; Santa Cruz Biotechnology) and mouse anti- gp130 (1:100; R&D Systems).

Techniques: Activity Assay, Injection, Transfection, Luciferase, Control, Chromatin Immunoprecipitation, Small Interfering RNA

Journal: Stroke

Article Title: Neuroprotection by Interleukin-6 Is Mediated by Signal Transducer and Activator of Transcription 3 and Antioxidative Signaling in Ischemic Stroke

doi: 10.1161/strokeaha.111.626648

Figure Lengend Snippet: Figure 5. Knockdown of IL-6R induces cell death in cerebral cortical neurons and enhancement of IL-6R by IL-6 reduces infarct volumes in cerebral ischemic injury. A, Cell death assessed by LDH activity in a medium of cortical neurons trans- fected with IL-6R- or gp130-specific siRNA for 24 hours or treated with AG490 for 24 hours (n4 per group). Data are meanSEM *P0.05, **P0.001 versus control. B, LDH activity in a medium of cortical neurons transfected with IL-6R- or gp130-specific siRNA for 24 hours in SOD2 WT or SOD2/ KO mouse brains (n4 per group). Data are meanSEM *P0.05 versus control. C, 2,3,5-triphenyltetrazolium chloride staining for analysis of infarct volumes after 24 hours of reperfusion after MCAO with or without IL-6 injection in male mice. D, Summary graph showing the size of infarction volumes. *P0.05 (n9 per group). NS indicates not significant; OD, optical density; IL-6R, interleukin-6 receptor; IL-6, interleukin-6; LDH, lactic dehydroge- nase; siRNA, small interfering RNA; WT, wild-type; KO, knock- out; MCAO, middle cerebral artery occlusion.

Article Snippet: The primary STROKE/2011/626648 Supplemental Data / 3 antibodies we used were rabbit anti-IL-6R (1:100; Santa Cruz Biotechnology) and mouse anti- gp130 (1:100; R&D Systems).

Techniques: Knockdown, Activity Assay, Control, Transfection, Staining, Injection, Small Interfering RNA, Knock-Out