Journal: Nucleic acids research

Article Title: Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS.

doi: 10.1093/nar/gkad522

Figure Lengend Snippet: Figure 2. Mutation signature filtering enables high-fidelity DMS probing of G base-pairing status. ( A ) G-specific mutation spectrums for in-cell probed RMRP and RNase P generated by SSII (green), Marathon (blue), and TGIRT (orange). Rates are shown separately for base-paired (filled) and single- stranded (open) G nucleotides. The y axis has a linear scale < 10 −2 (indicated by thick line) and logarithmic scale for values > 10 −2 (thin line). ( B ) Mutation rates (top) and percentage of detected mutations (bottom) measured by Marathon MaP for naturally-occurring N 1 -G and N 7 -G modifications in untreated E. coli and human rRNA. ( C ) ROC curves for muta tion-signa tur e-filter ed G r eacti vities for in-cell probed RMRP and RNase P. Curv es generated without mutation filtering for each enzyme are shown in gr ay. ( D ) Aver age AUROC across all probed RNAs quantifying the ability of muta tion-signa tur e-filter ed DMS reactivities to discriminate pairing status at each nucleotide. The best performing enzyme, Marathon, is boxed. See Supplementary Table S1 for the complete list of probed RNAs.

Article Snippet: Reverse transcription Four different mutational profiling (MaP) re v erse transcription (RT) protocols were evaluated, which we refer to by the RT enzyme used: Superscript II (SSII) ( 8 ), MarathonRT (Marathon) ( 37 ), T GIRT-III (T GIRT) ( 36 ) and e volv ed HIV RT (eHIV) ( 40 ).

Techniques: Mutagenesis, Generated

Journal: Nucleic acids research

Article Title: Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS.

doi: 10.1093/nar/gkad522

Figure Lengend Snippet: Figure 4. Four-base DMS-MaP conveys more structural information than other probing strategies. ( A ) Normalized reactivity profiles for in-cell probed RMRP gi v en by SHAPE-MaP (top), traditional DMS-MaP (SSII; no muta tion-signa ture filtering) considering only A and C nucleotides (upper middle) or all nucleotides (lower middle), or four-base DMS-MaP (bottom). Base pairing interactions in the accepted structur e ar e shown using gray shading and ar cs. ( B ) Unpair ed / pair ed likelihood ratios for SHAPE and four-base DMS-MaP reactivities measured on cell-free probed E. coli 16S and 23S rRNA. A likelihood ratio of 1 indicates that a base has equal probability of being paired or unpaired gi v en the measured reactivity. Likelihood ratios are shown for each nucleotide for four-base DMS-MaP and are aggregated for all nucleotides for SHAPE. ( C ) Comparison of AUROC (top) and expected structural information (ESI; bottom) for four-base DMS-MaP and SHAPE-MaP across a di v erse panel of RNAs. 2A3 SHAPE data for human rRNAs, and E. coli rRNAs and tmRNA were taken from ( 34 ). AUROC and ESI values were also calculated for only A and C DMS r eactivities, r epr esenting what is obtained by traditional two-base DMS probing experiments.

Article Snippet: Reverse transcription Four different mutational profiling (MaP) re v erse transcription (RT) protocols were evaluated, which we refer to by the RT enzyme used: Superscript II (SSII) ( 8 ), MarathonRT (Marathon) ( 37 ), T GIRT-III (T GIRT) ( 36 ) and e volv ed HIV RT (eHIV) ( 40 ).

Techniques: Comparison

Journal: Disease Markers

Article Title: The Key Genes Underlying Pathophysiology Association between Plaque Instability and Progression of Myocardial Infarction

doi: 10.1155/2021/4300406

Figure Lengend Snippet: Validations of screened hub genes. The protein expression levels of left ventricle at 1 d, 1 w, and 8 w after MI utilizing ELISA, including PAK3 (a), RASD2 (b), IQGAP2 (c), SLIT2 (d), CD44 (e), CD5 (f), SOCS3 (g), PIK3CD (h), VAV1 (i), and P2RY1 (j).

Article Snippet: For further validation, the left ventricular sample levels, including IQGAP2, Pak3, Slit2, CD44, CD5, SOCS3, and P2RY1, were measured using a mouse IQGAP2 ELISA kit (ybE288Mu, Ambion, China), mouse Pak3 ELISA kit (CSB-PA017407LA01HU, CUSABIO), mouse Slit2 ELISA kit (Q9R1B9, RayBiotech), mouse CD44 ELISA kit (P15379, RayBiotech), mouse CD5 ELISA kit (Q91X69, RayBiotech), mouse SOCS3 ELISA kit (EKU07517, BIOMATIK), mouse Vav1 ELISA kit (JN-S-86921, AFZHAN), and mouse P2RY1 ELISA kit (CSB-EL017326MO, CUSABIO), respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Disease Markers

Article Title: The Key Genes Underlying Pathophysiology Association between Plaque Instability and Progression of Myocardial Infarction

doi: 10.1155/2021/4300406

Figure Lengend Snippet: The correlation analysis of screened hub genes and LVEF. (a, b) The LVEF (a) and LVFS (b) of left ventricle at 1 d, 1 w, and 8 w after MI. (c–h) The expression levels of proteins, including PAK3 (c), CD44 (d), CD5 (e), SOCS3 (f), VAV1 (g), and PIK3CD (h) were demonstrated to be negatively correlated with LVEF. (i) The expression levels of protein P2RY1 were demonstrated to be positively correlated with LVEF.

Article Snippet: For further validation, the left ventricular sample levels, including IQGAP2, Pak3, Slit2, CD44, CD5, SOCS3, and P2RY1, were measured using a mouse IQGAP2 ELISA kit (ybE288Mu, Ambion, China), mouse Pak3 ELISA kit (CSB-PA017407LA01HU, CUSABIO), mouse Slit2 ELISA kit (Q9R1B9, RayBiotech), mouse CD44 ELISA kit (P15379, RayBiotech), mouse CD5 ELISA kit (Q91X69, RayBiotech), mouse SOCS3 ELISA kit (EKU07517, BIOMATIK), mouse Vav1 ELISA kit (JN-S-86921, AFZHAN), and mouse P2RY1 ELISA kit (CSB-EL017326MO, CUSABIO), respectively.

Techniques: Expressing

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: Acute liver failure occurrence reduced intestinal and hepatic soyasaponin II levels in mice. C57BL/6J mice were intraperitoneally administrated with D-galactosamine (700 mg/kg) in combination with lipopolysaccharide (10 μg/kg). Cecal contents and liver were collected after LPS/GalN treatment for 6 h. (A) Soyasaponin II level in cecal content and the representative chromatogram (n=5). (B) Soyasaponin II levels in liver and the representative chromatogram (n=3 for LPS/GalN-treated and n=4 for PBS-treated mice). *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques:

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: Soyasaponin II pretreatment mitigated acute liver failure. After pretreatment with or without soyasaponin II (5 mg/kg) for 3 days, mice were intraperitoneally administered D-galactosamine (700 mg/kg) in combination with lipopolysaccharide (10 μg/kg) for 6 h. (A) Effect of SS II (5 mg/kg, i.g) pretreatment on ALT levels (n=14-15 for LPS/GalN-treated and n=4 for PBS-treated mice). (B) Representative photographs of livers. (C) HE staining and (D) TUNEL staining of liver samples. The number of apoptotic cells was quantified (n=5 for LPS/GalN-treated and n=4 for PBS-treated mice). (E) qPCR analyses of hepatic Il-1β, Il-6, Cxcl-2, Ccl-2 gene expression. (n=8 for LPS/GalN treated and n=4 for PBS treated mice). (F) Immunohistochemical staining for CD11b in liver sections (n=5-7 for LPS/GalN-treated and n=3 for PBS-treated mice). (G) Level of serum Il-1β (n=8-9 for LPS/GalN treated and n=4 for PBS treated mice). Scale bars: 100 μm. *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques: Staining, TUNEL Assay, Gene Expression, Immunohistochemical staining

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: Soyasaponin II ameliorated canonical Nlrp3 inflammasome-associated innate immune response. After pretreatment with or without soyasaponin II (5 mg/kg) for 3 days, mice were intraperitoneally administered D-galactosamine (700 mg/kg) in combination with lipopolysaccharide (10 μg/kg) for 6 h. Hepatic macrophages were isolated, followed by transcriptome analysis. (A) Scatter plots of PCA of hepatic macrophage gene expression profile (n=3). (B) KEGG signaling pathway by GSEA (n=3). (C) Hub gene analysis of NOD-like receptor signaling pathway by CytoHubba (n=3). (D) Mature BMDMs were stimulated with DPBS or LPS for 2 h, with or without SSII (5 μg/ml) co-incubation. The relative mRNA levels of Nlrp3 and Il-1β (n=4). (E) Representative western blot analysis for Nlrp3, caspase-1 (precursor and spliced variant) and pro-Il-1β from BMDMs cell lysis solution and the quantification results (n=4). (F) Representative immunofluorescence images of ASC in BMDMs, and the fluorescence quantification results. Red represents ASC; Blue represents DAPI (n=3). (G) Cleaved Il-1β released in BMDMs culture supernatant was quantified by ELISA (n=10 for LPS-treated and n=8 for PBS-treated cells) and Il-1β levels from isolated liver macrophages (n=4-5 for LPS/GalN-treated and n=3 for PBS-treated mice). Scale bars: 20 μm. *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques: Isolation, Gene Expression, Incubation, Western Blot, Variant Assay, Lysis, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: Soyasaponin II blocked YB-1 activation in macrophages. (A, B) Heat-Map and volcano plots of liver macrophages proteins with protein cluster analysis of differentially regulated proteins in DMSO and SSII groups from LPS/GaIN treated animals. Plot of SSII/DMSO ratios (Log 2 FC) versus -Log 10 FDR of liver macrophage proteins: green dots represent downregulated proteins, red dots represent differentially up-regulated proteins. (C) Protein-protein interaction (PPI) network diagram of discrepant proteins connected with YB-1 from the two groups. (D) Mature BMDMs were stimulated with PBS or LPS for 2 h, with or without co-incubation of SSII (5 μg/ml), and cells harvested after activation with ATP (5mM) for 30min. Representative western blot analysis for phosphorylated YB-1 and total YB-1 in cell lysis solution is displayed, as well as the protein quantification (n=4). (E) Left panel: PBS or LPS primed BMDMs with (n=3) or without (n=3) co-incubation with SSII (5 μg/ml) in petri dishes for 2 h. Immunofluorescence staining with p-YB-1 antibody and the fluorescence quantification. Right panel: Macrophages isolated from LPS/GaIN treated mouse liver with (n=3) or without (n=3) SSII pretreatment for three days. Immunofluorescence staining with p-YB-1 antibody and the fluorescence quantification. (F) BMDMs were stimulated with PBS or LPS for 15 min, with or without co-incubation of SSII (5 μg/ml). Representative western blot for phosphorylated AKT in BMDMs and the quantification results (n=3). (G) BMDMs was pretreated with LY294002 (10 μM) for 30 min, then were stimulated with LPS co-treatment with or without soyasaponin II. Representative western blot for phosphorylated YB-1 and the quantification results (n=3). Scale bars: 10 μm. *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques: Activation Assay, Incubation, Western Blot, Lysis, Immunofluorescence, Staining, Fluorescence, Isolation

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: The repression of Nlrp3 inflammasome by soyasaponin II was dependent on YB-1. (A) BMDMs were transfected with YB-1 si-RNA plasmid or negative control (NC) plasmid for 36 h, then incubation with LPS or PBS for 2 h. The relative mRNA levels of Nlrp3 (n=12 for LPS-treated and n=4 for PBS-treated cells) and Il-1β (n=12-13 for LPS-treated and n=8-9 for PBS-treated cells). (B) BMDMs were transfected with pcDNA-YB-1 mutant (Ser102 to Glu102, p-YB-1 overexpression) plasmid or pcDNA-YB-1 (control) plasmid for 36 h, followed by LPS treatment for 2 h. The relative mRNA levels of Nlrp3 and Il-1β mRNAs (n=8). (C) BMDMs were transfected with YB-1 si-RNA plasmid or negative control (NC) plasmid for 36 h, then incubation with LPS or PBS for 2 h. Western blot of Nlrp3, pro-caspase-1, caspase-1 p20 and pro-Il-1β, as well as the quantification results (n=3). (D) Cleaved Il-1β released in cell supernatant were quantified by ELISA (PBS group n=4, LPS group n=20). (E) LPS-primed BMDMs were co-incubated with or without SSII for 2 h, transfected with or without YB-1 si-RNA for 36 h, and the relative expression mRNA levels of Nlrp3 (n=5 for YB-1 si-RNA-treated and n=6-10 for negative control (NC) plasmid -treated cells) and Il-1β (n=7). (F) Western blot of Nlrp3, pro-caspase-1, caspase-1 p20 and pro-Il-1β, as well as the quantification results (n=5). (G) Cleaved Il-1β released in cell supernatant were quantified by ELISA (n=8). *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Mutagenesis, Over Expression, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Theranostics

Article Title: Soyasaponin II protects against acute liver failure through diminishing YB-1 phosphorylation and Nlrp3-inflammasome priming in mice

doi: 10.7150/thno.40128

Figure Lengend Snippet: Phosphorylated YB-1 was enriched in the macrophage nuclei of ALF patients. (A, B) Macrophages in human liver paraffin section marked by CD64 antibody, and p-YB-1 expression were detected by immunofluorescence, and the p-YB-1 nuclear translocation positive macrophages were quantified (control n=5; ALF n=11). (C) Working model: LPS/GalN challenge decreased fecal and hepatic soyasaponin II levels. Soyasaponin II repressed YB-1 phosphorylation and nuclear translocation, p-YB-1 could translocate into nuclear and bind the promoter of Nlrp3, to promote Nlrp3 inflammasome priming, Il-1β overproduction and liver damage. Scale bars: 10 μm. *p<0.05.

Article Snippet: Mice were given oral administration of soyasaponin II (SSII; ChromaDex, 5 mg/kg dissolved in dimethyl sulfoxide (DMSO)) once a day for three consecutive days.

Techniques: Paraffin Section, Expressing, Immunofluorescence, Translocation Assay, Control, Phospho-proteomics

Journal: Cell Reports Medicine

Article Title: The adipose-neural axis is involved in epicardial adipose tissue-related cardiac arrhythmias

doi: 10.1016/j.xcrm.2024.101559

Figure Lengend Snippet: Leptin secreted by adipocytes causes an irregular rhythm of cardiomyocytes by activating sympathetic neurons via the leptin receptor (A) Quantification of cytokines in the adipocyte supernatant using a bead-based multiplex assay ( n = 3 times per group). (B) Line-scan images of spontaneous Ca 2+ transients in hiPSC-CMs treated with IL-1β or IL-6 with or without hiPSC-SymNs. (C) Line-scan images of spontaneous Ca 2+ transients in hiPSC-CMs treated with IL-8 or TNF-α with or without hiPSC-SymNs. (D) Quantification of the beating rate and cells exhibiting irregular Ca 2+ transients in hiPSC-CMs cocultured with sympathetic neurons and a single cytokine ( n = 60 cells per group). Data are presented as the mean ± SEM. ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, determined by Student’s t test. (E) Line-scan images of spontaneous Ca 2+ transients and quantification of the beating rate and cells exhibiting irregular Ca 2+ transients in hiPSC-CMs treated with Lep with or without hipSC-SymNs ( n = 60 cells per group). Data are presented as the mean ± SEM. ∗∗∗ p < 0.001, determined by Student’s t test. (F) Line-scan images of spontaneous Ca 2+ transients and quantification of the beating rate and cells exhibiting irregular Ca 2+ transients in hiPSC-CMs treated with APN with or without hiPSC-SymNs ( n = 60 cells per group). Data are presented as the mean ± SEM. ns, not significant; ∗∗ p < 0.01, determined by Student’s t test. (G) Immunofluorescence staining for the sympathetic neuron marker TH and the leptin receptor (LepR). Scale bar, 50 μm. (H) Immunofluorescence assay for TH and the neuronal activity marker c-Fos in hiPSC-SymNs cultured with or without leptin. Scale bar, 50 μm. (I) The concentrations of neurotransmitters in the supernatant were analyzed using commercial ELISA kits before and after leptin administration ( n = 3 times per group). (J) Western blot analysis and relative quantification of STAT3, p-STAT3, and SOCS3 in hiPSC-SymNs treated with leptin, adi sup, or adi sup plus a JAK2 inhibitor (AG490) ( n = 3 times per group). (K) Line-scan images of spontaneous Ca 2+ transients and quantification of cells exhibiting irregular Ca 2+ transients in hiPSC-CMs from the triple coculture system with the addition of a leptin-neutralizing antibody ( n = 60 cells per group). Data are presented as the mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, determined by one-way ANOVA test.

Article Snippet: SOCS3 (D6E1T) Rabbit mAb , Cell Signaling Technology , Cat# 52113; RRID: AB_2799408.

Techniques: Multiplex Assay, Immunofluorescence, Staining, Marker, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: The adipose-neural axis is involved in epicardial adipose tissue-related cardiac arrhythmias

doi: 10.1016/j.xcrm.2024.101559

Figure Lengend Snippet:

Article Snippet: SOCS3 (D6E1T) Rabbit mAb , Cell Signaling Technology , Cat# 52113; RRID: AB_2799408.

Techniques: Recombinant, Clinical Proteomics, Derivative Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Western Blot, Software

Journal: JCI Insight

Article Title: Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

doi: 10.1172/jci.insight.131340

Figure Lengend Snippet: (A) Schematic depiction of BALF processing for SOCS3 ELISA. (B) Levels of secreted SOCS3 in patient BALF. Data points represent individual patients; horizontal bars represent mean values (n = 12-22 patients). Two groups are still significantly different even if 3 normal subjects with highest SOCS3 levels are excluded. Students 2-tailed t test was used for comparison of 2 groups; *P < 0.05.

Article Snippet: Samples were sonicated to disrupt EVs and were analyzed using a human or rat SOCS3 ELISA kit (Cloud-Clone Corp.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

doi: 10.1172/jci.insight.131340

Figure Lengend Snippet: (A) Experimental scheme: WT and LSL-KrasG12D mice were inoculated oropharyngeally with adenoviral Cre recombinase on day 0. BALF was harvested at 4, 8, 12, and 16 weeks after tumor initiation and analyzed for secreted SOCS3 as well as cell counts; alternatively, AMs were purified by adherence for analysis of their CM. Representative histopathologic lesions from mice harvested at the indicated time points are shown. AAH, atypical adenomatous hyperplasia. (B) SOCS3 concentrations as determined by ELISA in BALF harvested at indicated time points from WT and G12D mice (n = 3–7 mice per group; each symbol represents a value from an individual mouse). (C) Representative WB for SOCS3 of BALF samples collected from WT and Kras mutant mice at 4, 8, 12, and 16 weeks after tumor initiation. Each lane represents a sample from an individual mouse (n = 2 mice). Lanes on either side of the dashed line were from the same blot but were noncontiguous. (D) Numbers of total lavageable cells in the BALF of WT and Kras mutant mice at varying times after tumor initiation (n = 3 mice). (E) Numbers of resident AMs (defined as CD11c+CD11b–Siglec-F+ cells) in the BALF of WT and Kras mutant mice at various times after tumor initiation (n = 2 groups of cells pooled from 2 individual mice). (F) At 10 weeks after tumor initiation with SPC (alveolar EC targeted) specific adenoviral Cre recombinase, BALF was harvested and analyzed for SOCS3 by ELISA (n = 3–4 mice per group). (G) Concentration of SOCS3 in CM of AMs isolated from WT and Kras mutant mice at the indicated times after tumor initiation. Cells isolated from the BALF of individual mice of each strain (4 mice per group) were pooled and then divided into triplicate samples. Cells were plated at a concentration of 1 × 106 cells/ml for ~20 h. The resulting CM was sonicated and analyzed for SOCS3 by ELISA; data are mean of 3–4 samples. (H) Relative SOCS3 protein levels determined by WB of lysates of AMs obtained at 16 weeks after tumor initiation; note that these are from the same cell cultures whose CM was utilized for ELISA determination in G (n = 3). (I) EV numbers determined in AM CM via flow cytometric analysis and expressed as EV number per 1 × 106 AMs; data are mean of triplicate samples. Student’s 2-tailed t test was used for comparison of 2 groups (F and H), or 1-way ANOVA with Bonferroni’s post hoc analysis was used for multiple comparisons (B, D, E, G, and I); *P < 0.05.

Article Snippet: Samples were sonicated to disrupt EVs and were analyzed using a human or rat SOCS3 ELISA kit (Cloud-Clone Corp.).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Mutagenesis, Concentration Assay, Isolation, Sonication

Journal: JCI Insight

Article Title: Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

doi: 10.1172/jci.insight.131340

Figure Lengend Snippet: (A) Schematic of liposome generation. (B) Histogram: Size distribution of both empty and SOCS3-containing liposomes from 1 representative liposome preparation. (C) Representative WB comparing relative SOCS3 protein amounts in EVs isolated from 1 × 106 AMs vs. serial dilutions of synthetic SOCS3-containing liposomes. (D) A549 cells were pretreated with liposomes containing 10 ng of SOCS3 protein, or an equal volume of empty liposomes containing only PBS, for 1 h. They were then washed and stimulated with 10 ng/ml IL-6 for 30 minutes, and lysates were probed for pSTAT3. Top: Representative WB; bottom: mean densitometric analysis of 3 separate experiments, represented as relative pSTAT3 protein. Dashed line denotes noncontiguous lanes from the same blot. pSTAT3 and total STAT3 were from separate gels/blots. Student’s 2-tailed t test was used for comparison of 2 groups; *P < 0.05.

Article Snippet: Samples were sonicated to disrupt EVs and were analyzed using a human or rat SOCS3 ELISA kit (Cloud-Clone Corp.).

Techniques: Isolation

Journal: JCI Insight

Article Title: Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

doi: 10.1172/jci.insight.131340

Figure Lengend Snippet: (A) A549 cells were incubated with liposomes containing 10 ng SOCS3 or an equal volume of liposomes containing PBS alone (empty) for 72 h and analyzed by CyQuant cell proliferation assay measuring total cellular DNA (n = 3 separate experiments). (B) One thousand A549 cells were plated per well of a 96-well tissue culture plate and were incubated with empty or SOCS3 liposomes for 72 h. Cellular confluence was measured using an IncuCyte Live Cell Analysis system. Data points represent the mean of quadruplicate measurements of confluency, expressed as AU per well, and are representative of triplicate wells per condition. (C) A549 cells were treated with empty or SOCS3 liposomes for 24 h and analyzed for FoxM1 mRNA expression by qPCR (n = 3 separate experiments). (D) Representative micrographs of caspase-3/7–positive cells after liposome treatment. Concurrent with the confluence assay in B, A549 cells were incubated with IncuCyte caspase-3/7 apoptosis assay reagent and empty or SOCS3 liposomes. Fluorescence micrographs are representative of 3 triplicate wells per treatment. Scale bar: 100 μm. (E) A549 cells were treated with empty or SOCS3 liposomes or FasL (as a positive control) for 24 h, and annexin V+ cells determined via flow cytometry (n = 4–5 replicate samples). (F) A549 cells were treated with empty or SOCS3 liposomes for 24 h and analyzed for relative FasR mRNA expression by qPCR (n = 4 separate experiments). (G) A549 cells were treated with FasL, empty liposomes, or SOCS3 liposomes for 24 h, with or without the pan-caspase inhibitor Z-VAD-FMK (Z), and cell proliferation was determined by CyQuant assay (n = 5–14 replicates from 3 separate experiments). (H) Top: Schematic depiction of CSE transformation protocol for WT or G12V-RLE cells. A total of 0.5 × 106 RLE-6TN cells were incubated with either WT or G12V retrovirus for 24 h. Cells were washed and selected with puromycin for an additional 48–72 h. Surviving cells were passaged an additional 2 times before use in experiments. For CSE transformation, cells were plated at a concentration of 10,000 cells/well of a 6-well tissue culture dish and treated with 3% CSE, along with empty or SOCS3 liposomes, for 1 h daily for 7 consecutive days. They were then cultured in equal numbers in a soft agar colony assay for an additional 7 days before colonies were enumerated. Bottom left: Representative micrographs of CSE-treated WT or G12V RLEs within a soft agar colony assay. Photographs were taken at 40× magnification. Scale bar: 1000 μm. Bottom right: Data represents mean colony counts from 7 separate 40× fields per treatment from 3 separate experiments. Student’s 2-tailed t test was used for comparison of 2 groups (A, C, F, and H), or 1-way ANOVA with Bonferroni’s post hoc analysis was used for multiple comparisons (E and G); *P < 0.05 or #P < 0.05 represents comparison to control or empty liposomes, respectively.

Article Snippet: Samples were sonicated to disrupt EVs and were analyzed using a human or rat SOCS3 ELISA kit (Cloud-Clone Corp.).

Techniques: Incubation, CyQUANT Assay, Proliferation Assay, Expressing, Apoptosis Assay, Fluorescence, Positive Control, Flow Cytometry, Transformation Assay, Concentration Assay, Cell Culture, Colony Assay

Journal: JCI Insight

Article Title: Alveolar macrophage secretion of vesicular SOCS3 represents a platform for lung cancer therapeutics

doi: 10.1172/jci.insight.131340

Figure Lengend Snippet: (A) Experimental scheme: 1 × 106 A549 cells were injected s.c. in athymic nude mice at day 0. After palpable tumors were observed at ~28 days, mice were randomized into 2 groups and treated s.c. with liposomes containing 10 ng SOCS3 or an equal volume of liposomes containing PBS (at 2 sites per tumor) in 50 µl PBS approximately every other day for a total of 6 treatments. Tumor measurements were taken 2 days prior to the first liposome injection and on every subsequent injection day. (B) Mean tumor volumes from empty and SOCS3 treated mice (n = 4–7 mice per group). (C) Images of representative whole tumors from mice treated with empty and SOCS3 liposomes. (D) Representative images of isotype, pSTAT3 and DAPI labeled tumors sections. Solid arrows represent nuclear-associated pSTAT3; open arrowheads represent cytoplasmic-associated pSTAT3. (E) Representative H&E-stained tumors from A549 tumor–bearing mice treated with empty and SOCS3 liposomes. One-way ANOVA with Bonferroni’s post hoc analysis was used for multiple comparisons.

Article Snippet: Samples were sonicated to disrupt EVs and were analyzed using a human or rat SOCS3 ELISA kit (Cloud-Clone Corp.).

Techniques: Injection, Labeling, Staining

Journal: Cell Reports

Article Title: A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory gene expression

doi: 10.1016/j.celrep.2025.115530

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were: anti-FLAG M2 (Sigma F3165, RRID: AB_259529 ), anti-pY705-STAT3 clone D3A7 (CST #9145, RRID: AB_2491009 ), anti-STAT3 clone 124H6 (CST #9139, RRID: AB_331757 ), anti-SOCS3 polyclonal (Proteintech #14025-1-AP, RRID: AB_10597854 ), anti-GSK3β clone D5C5Z (CST #12456, RRID: AB_2636978 ) and anti-pTyr-1000 monoclonal pool (CST #8954, RRID: AB_2687925 .

Techniques: Virus, Recombinant, Phospho-proteomics, Mutagenesis, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Genome Wide, Mass Spectrometry, Cell Culture, Software

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3 is induced in podocytes in the imiquimod-induced lupus model. (A) BALB/c mice were treated with or without topical imiquimod for 4 weeks. Glomeruli were isolated by the Dynabeads perfusion method. The expression levels of SOCS family members in the glomeruli were evaluated by qPCR analysis. Data are means±SEM for eight mice in each group. Data are compiled from two independent experiments. *p<0.05. (B) Conditionally immortalised murine podocytes were stimulated with or without IL-6 for 2 hours, and the expression levels of SOCS3 were evaluated by qPCR analysis. Data are compiled of four independent experiments. ***p<0.001. (C) Mice lacking SOCS3 expression in podocytes (podocin-Cre × SOCS3 fl/fl mice (SOCS3-cKO mice)) and SOCS3-sufficient mice (SOCS3 fl/fl mice) (SOCS3-WT mice) were treated with or without topical imiquimod for 4 weeks. Glomeruli were isolated and the expression levels of SOCS3 in the glomeruli were evaluated by qPCR analysis. Data are means±SEM for eight to nine mice in each group. Data are compiled from two independent experiments. ***p<0.001. CISH, cytokine-inducible SH2-containing protein; IMQ, imiquimod; KO, knockout; ns, not significant; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Isolation, Expressing, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3-cKO mice exhibit severe glomerulonephritis in the imiquimod-induced lupus model. SOCS3-cKO mice and SOCS3-WT mice were treated with or without topical imiquimod as described in the Methods section. (A) Representative photomicrograph of periodic acid-Schiff (PAS)-stained kidney sections from SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. (B) Renal histopathologic scores in SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. Data are means±SEM for 10 mice in each group. Data are compiled from two independent experiments. *p<0.05. (C) Serum creatinine levels in SOCS3-cKO mice and SOCS3-WT mice at 12 weeks. Data are means±SEM for 10–12 mice in each group. Data are compiled from two independent experiments. **p<0.01. (D) Urine albumin levels in SOCS3-cKO mice and SOCS3-WT mice at 12 weeks. Data are means±SEM for 8–9 mice in each group. Data are compiled from two independent experiments. *p<0.05. (E) Renal sections of SOCS3-cKO mice and SOCS3-WT mice at 8 weeks were stained with anti-IgM antibody. Shown are representative photographs. (F) The frequencies of the glomeruli having IgM deposits in 20 glomeruli were evaluated. Data are means±SEM for 10 mice in each group. Data are compiled from two independent experiments. ***p<0.001. IMQ, imiquimod; KO, knockout; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Staining, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: Autoantibody production is increased in SOCS3-cKO mice. SOCS3-cKO mice and SOCS3-WT mice were treated with or without topical imiquimod as described in the Methods section. (A) Anti-dsDNA antibody levels in sera in SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. Data are means±SEM for 11 mice in each group. Data are compiled from two independent experiments. *p<0.05. (B) Shown are representative dot plots of CXCR5 vs PD-1 gated on CD4 + T cells (left) and frequency of Tfh cells (CXCR5 + PD-1 + cells) (right) in spleen in SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. Data are means±SEM for 11 mice in each group. Data are compiled from two independent experiments. *p<0.05. (C) Shown are representative dot plots of Fas vs GL7 gated on B220 + B cells (left) and frequency of GC B cells (Fas + GL7 + cells) (right) in spleen in SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. Data are means±SEM for 11 mice in each group. Data are compiled from two independent experiments. ***p<0.001. (D) Representative photographs of spleen sections stained with PNA at 8 weeks. (E) Shown are means±SEM of average GC areas (square millimetres × 10 4 ) (left) and of the number of GC formation (per square millimetre) (right). Data are means±SEM for 10 mice in each group. Data are compiled from two independent experiments. *p<0.05. Anti-dsDNA, anti-double-strand DNA; GC, germinal centre; IMQ, imiquimod; KO, knockout; PD-1, programmed cell death-1; PNA, peanut agglutinin; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Staining, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3 expression in CD4 + T cells, CD8 + T cells and B cells in SOCS3-cKO mice. Splenocytes were obtained from 8-week-old SOCS3-cKO mice and SOCS3-WT mice. (A) CD4 + T cells, (B) CD8 + T cells and (C) B cells were isolated as described in the Methods section, and were stimulated with or without IL-6 for 2 hours. The expression levels of SOCS3 were evaluated by qPCR analysis. Data are compiled of four independent experiments. IL-6, interleukin 6; KO, knockout; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Expressing, Isolation, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: NP-OVA-induced antibody production in SOCS3-cKO mice. Eight-week-old SOCS3-cKO mice and SOCS3-WT mice were immunised intraperitoneally with NP-OVA as described in the Methods section. Seven days after the immunisation, serum levels of NP-specific IgG1 (A) and IgG2a (B) were quantified by ELISA. Data are means±SEM for six mice in each group. Data are compiled from two independent experiments. IgG, immunoglobulin G; KO, knockout; NP-OVA, 4-hydroxy-. 3- nitrophenylacetyl-ovalbumin; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: CRISPR-Cas9-mediated gene deletion of SOCS3 in a murine podocyte cell line was performed as described in the Methods section. Cells were stimulated with or without IL-6 for 2 hours and were stained with anti-SOCS3 antibody and DAPI. Shown are representative photographs. DAPI, 4',6-diamidino-2-phenylindole; DIC, differential interference contrast; IL-6, interleukin 6; KO, knockout; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: CRISPR, Staining, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3 expressed in podocytes suppresses local and systemic IL-6 expression. (A) CRISPR-Cas9-mediated gene deletion of SOCS3 in a murine podocyte cell line was performed as described in the Methods section. Cells were stimulated with or without IL-6 for 2 hours, and the expression levels of indicated genes were evaluated by qPCR analysis. Data are compiled of four independent experiments. *p<0.05. (B) SOCS3-cKO mice and SOCS3-WT mice were treated with or without topical imiquimod for 8 weeks as described in the Methods section. Glomeruli were isolated and the expression levels of indicated genes in the glomeruli were evaluated by qPCR analysis. Data are means±SEM for eight mice in each group. Data are compiled from two independent experiments. *p<0.05. (C) Serum levels of IL-6 in SOCS3-cKO mice and SOCS3-WT mice at 8 weeks. Data are means±SEM for eight mice in each group. Data are compiled from two independent experiments. *p<0.05. CXCL, chemokine (C-X-C motif) ligand; GM-CSF, granulocyte macrophage colony-stimulating factor; IL6, interleukin 6; IMQ, imiquimod; KO, knockout; SOCS, suppressor of cytokine signalling; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Expressing, CRISPR, Isolation, Knock-Out

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3 and IL-6 are expressed in podocytes in MRL/lpr mice. Single-cell suspensions were prepared from isolated glomeruli in 6-month-old MRL/lpr mice and control MRL/+mice. Intracellular staining of SOCS3 and IL-6, together with staining of podocin (NPHS2), was performed as described in the Methods section. (A) Left panels are the representative SOCS3 staining (red) and isotype-matched control staining (grey) gated on NPHS2 + cells in control MRL/+mice and MRL/lpr mice and on NPHS2 − cells in MRL/lpr mice. Data are means±SEM of mean fluorescence intensity (MFI) of SOCS3 for eight mice in each group. Data are compiled from two independent experiments. ***p<0.001, **p<0.01. (B) Left panels are the representative IL-6 staining (red) and isotype-matched control staining (grey) gated on NPHS2 + cells in control MRL/+mice and MRL/lpr mice and on NPHS2 − cells in MRL/lpr mice. Data are means±SEM of MFI of IL-6 for eight mice in each group. Data are compiled from two independent experiments. ***p<0.001, **p<0.01. IL-6, interleukin 6; SOCS, suppressor of cytokine signalling.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Isolation, Control, Staining, Fluorescence

Journal: Lupus Science & Medicine

Article Title: Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model

doi: 10.1136/lupus-2020-000426

Figure Lengend Snippet: SOCS3-cKO mice exhibit reduced survival rates in the imiquimod-induced lupus model. SOCS3-cKO mice and SOCS3-WT mice were treated with topical imiquimod as described in the Methods section. Shown are cumulative survival rates of SOCS3-cKO mice and SOCS3-WT mice following topical administration of imiquimod on the ear skin three times per week, n=12, each. KO, knockout; SOCS, suppressor of cytokine signalling; WT, wild type.

Article Snippet: Murine podocyte cell lines were immunostained with FITC-conjugated rabbit anti-mouse SOCS3 antibody (Biorbyt LLC, San Francisco, California, USA).

Techniques: Knock-Out

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Wnt5a skews dendritic cell differentiation to an unconventional phenotype with tolerogenic features.

doi: 10.4049/jimmunol.1101243

Figure Lengend Snippet: FIGURE 4. Changes in the transcription profile of Wnt5a-DCs correlate with their unconventional phenotype. A and B, Real-time PCR quantification of mRNA levels in day 6 iDCs differentiated from monocytes in the presence or absence of Wnt proteins. mRNA expression of different TLRs (A) and transcriptional factors and regulatory proteins (B) in Wnt-DCs are expressed relative to individual control values (black bars; referred to as 1). C, Control and Wnt5a-DCs were stimulated with LPS for 3 h, and IRF1 and IRF8 mRNA expression was analyzed. Variations between samples were corrected by normalizing against the expression of GAPDH, which was used as an endogenous control. Data represent the mean (6SD) of three to five independent experiments. D and E, Intracellular IRF1, IRF8 (D), ID3, and SOCS3 (E) protein levels were analyzed by flow cytometry in control and Wnt5a day 6 iDCs or after 20 h of LPS stimulation. The total MFI (in parentheses) is shown in each histogram. Data are representative of three independent experiments. *p # 0.05, **p # 0.005, ***p # 0.0005, by unpaired t test.

Article Snippet: For the intracellular staining of IFN regulatory factor 1 (IRF1), IRF8, inhibitor of DNA binding 3 (ID3), suppressors of cytokine signaling 3 (SOCS3), and total b-catenin, cells were fixed and permeabilized using Cytofix/ Cytoperm solution, washed with Perm/Wash buffer (BD Biosciences), and stained with specific rabbit polyclonal Abs for IRF1, IRF8, ID3, and SOCS3 (all from Santa Cruz Biotechnology) followed by allophycocyanin-conjugated donkey anti-rabbit IgG or with FITC-conjugated anti-b-catenin mAb (BD Biosciences) (14).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Wnt5a skews dendritic cell differentiation to an unconventional phenotype with tolerogenic features.

doi: 10.4049/jimmunol.1101243

Figure Lengend Snippet: FIGURE 5. Wnt5a interferes with GM-CSF/IL-4 cytokine signaling and modified the early DC differentiation program. Human CD14+ monocytes were cultured with GM-CSF/IL-4 in either the presence or the absence of Wnt3a or Wnt5a. A, After 2 or 20 h of culture, intracellular STAT5 and STAT6 tyrosine phosphorylation levels were analyzed by flow cytometry. The percentage of positive cells, determined according to background staining (gray histograms), and the total MFI are show in each histogram. Data are representative of four independent experiments. B, SOCS1 and SOCS3 expression was analyzed by quantitative RT-PCR analysis during Wnt-DC differentiation (3 and 20 h of culture and day 6 iDCs). Data shown are relative to individual control values (black; set as 1). C, Monocytes were cultured as indicated for 0 (monocyte, Mo), 3, or 20 h, and the expression of several transcription factors and regulatory proteins were analyzed by quantitative RT-PCR. Line graphs represent the kinetics of mRNA expression in control (black squares), Wnt3a (open circles), or Wnt5a (open triangles) cultures; data are representative of three independent experiments. Bar graphs show transcription levels relative to individual control values (black bar; referred to as 1). Data represent the mean (6SD) of four independent experiments. Variations between samples were corrected by normalizing against the expression of GAPDH, which was used as an endogenous control. D–F. The first 24 h of culture are sufficient for inducing Wnt5a effects in DC differentiation. Human CD14+ monocytes were cultured in the GM-CSF/IL-4 cytokine mixture as described above. Wnt5a was added to the DC differentiation culture (Wnt5a-DCs) or during just the first 24 h (24 h-Wnt5a-DCs). To remove Wnt5a, culture medium was replaced with 24 h GM-CSF/IL-4 monocyte-conditioned medium. Both control and Wnt5a cultures underwent an equivalent process. D, Flow cytometry analysis of day 6 iDCs. The percentage of CD14+ cells and the mean fluorescence values of CD86 and PDL1 are shown for Wnt5a and 24 h-Wnt5a-DCs relative to those of control iDCs cultures. E, Control, Wnt5a, and 24 h-Wnt5a-DCs were stimulated with LPS for 48 h. Production of IL-12p70, TNF-a, and IL-10 in culture supernatants was quantified by ELISA. F, Cytokine production of primed T cells. DCs stimulated with LPS were cultured in MLR with naive CD4+

Article Snippet: For the intracellular staining of IFN regulatory factor 1 (IRF1), IRF8, inhibitor of DNA binding 3 (ID3), suppressors of cytokine signaling 3 (SOCS3), and total b-catenin, cells were fixed and permeabilized using Cytofix/ Cytoperm solution, washed with Perm/Wash buffer (BD Biosciences), and stained with specific rabbit polyclonal Abs for IRF1, IRF8, ID3, and SOCS3 (all from Santa Cruz Biotechnology) followed by allophycocyanin-conjugated donkey anti-rabbit IgG or with FITC-conjugated anti-b-catenin mAb (BD Biosciences) (14).

Techniques: Cell Culture, Phospho-proteomics, Cytometry, Staining, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay