anti gapdh  (Proteintech)

 
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  • 99
    Name:
    Mouse GAPDH Monoclonal
    Description:
    The GAPDH antibody from Proteintech is a mouse monoclonal antibody to a recombinant protein of human GAPDH This antibody recognizes human mouse rat zebrafish yeast plant antigen The GAPDH antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    Catalog Number:
    60004-1-Ig
    Price:
    [219.03]
    Applications:
    IHC,Western Blot,ELISA,Flow Cytometry,Immunoprecipitation,Immunofluorescence
    Host:
    Mouse
    Conjugate:
    Unconjugated
    Immunogen:
    Recombinant Protein
    Size:
    150 ul
    Category:
    Antibody
    Antibody Type:
    Primary antibody
    Isotype:
    IgG2b
    Reactivity:
    Human Mouse Pig Rat Zebrafish Yeast Monkey Xenopus Hamster Plant
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    Structured Review

    Proteintech anti gapdh
    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and <t>GAPDH)</t> in exosomes. <t>FASN</t> and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS
    The GAPDH antibody from Proteintech is a mouse monoclonal antibody to a recombinant protein of human GAPDH This antibody recognizes human mouse rat zebrafish yeast plant antigen The GAPDH antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    https://www.bioz.com/result/anti gapdh/product/Proteintech
    Average 99 stars, based on 144 article reviews
    Price from $9.99 to $1999.99
    anti gapdh - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells"

    Article Title: Proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells

    Journal: Journal of Ovarian Research

    doi: 10.1186/s13048-020-0609-y

    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS
    Figure Legend Snippet: Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS

    Techniques Used: Tandem Mass Spectroscopy, Western Blot, Marker, Negative Control, Derivative Assay

    2) Product Images from "Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma"

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    Journal: Clinical and Translational Medicine

    doi: 10.1002/ctm2.41

    The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.
    Figure Legend Snippet: The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.

    Techniques Used: RNA Sequencing Assay, Western Blot, Expressing, Isolation

    ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.
    Figure Legend Snippet: ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.

    Techniques Used: Western Blot, Expressing

    Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P
    Figure Legend Snippet: Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P

    Techniques Used: In Vitro, Western Blot, Expressing, Derivative Assay, Cell Culture, Immunofluorescence, Staining, CCK-8 Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Irradiation

    3) Product Images from "METTL3/YTHDF2 m6A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer, et al. METTL3/YTHDF2 m6A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer"

    Article Title: METTL3/YTHDF2 m6A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer, et al. METTL3/YTHDF2 m6A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15063

    METTL3 and YTHDF2 are highly expressed in BCa. A, B, Transcript levels of METTL3 and YTHDF2 in primary tumours vs normal subjects and mRNA expression levels among stages from TCGA database. C, Expression levels of METTL3 and YTHDF2 in superficial and infiltrating BCa from Oncomine online database. D, Western blot assay showing the up‐regulated expression levels of METTL3 and YTHDF2 in BCa cancer cell lines (T24, UM‐UC‐3) compared to the human normal urothelium cell line (SV‐HUC‐1). GAPDH was used for the normalization control. E, Pearson correlation analysis of METTL3 and YTHDF2 in primary tumours (n = 408) from TCGA database; r = .2036, P
    Figure Legend Snippet: METTL3 and YTHDF2 are highly expressed in BCa. A, B, Transcript levels of METTL3 and YTHDF2 in primary tumours vs normal subjects and mRNA expression levels among stages from TCGA database. C, Expression levels of METTL3 and YTHDF2 in superficial and infiltrating BCa from Oncomine online database. D, Western blot assay showing the up‐regulated expression levels of METTL3 and YTHDF2 in BCa cancer cell lines (T24, UM‐UC‐3) compared to the human normal urothelium cell line (SV‐HUC‐1). GAPDH was used for the normalization control. E, Pearson correlation analysis of METTL3 and YTHDF2 in primary tumours (n = 408) from TCGA database; r = .2036, P

    Techniques Used: Expressing, Western Blot

    YTHDF2 promotes the migration of cancer cells. A, Representative Western blots show the suppressed expression of YTHDF2 by the siRNA pool in BCa cell lines. GAPDH served as the normalization control. B, C, Wound healing assay (B) and trans‐well assay (C) demonstrating the inhibited migration ability upon YTHDF2 depletion in cancer cell lines. D, Representative Western blots showing the altered expression level of EMT‐associated proteins. GAPDH served as the normalization control
    Figure Legend Snippet: YTHDF2 promotes the migration of cancer cells. A, Representative Western blots show the suppressed expression of YTHDF2 by the siRNA pool in BCa cell lines. GAPDH served as the normalization control. B, C, Wound healing assay (B) and trans‐well assay (C) demonstrating the inhibited migration ability upon YTHDF2 depletion in cancer cell lines. D, Representative Western blots showing the altered expression level of EMT‐associated proteins. GAPDH served as the normalization control

    Techniques Used: Migration, Western Blot, Expressing, Wound Healing Assay

    4) Product Images from "Chromosome Segregation Is Biased by Kinetochore Size"

    Article Title: Chromosome Segregation Is Biased by Kinetochore Size

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.03.023

    Polar Ejection Forces on Chromosome Arms Ensure Mitotic Fidelity but Are Not Implicated in the Observed Missegregation Bias for Chromosomes with Large Kinetochores (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) in control (top) and Kif4a RNAi (bottom) cells treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Scale bar, 5 μm. Time, hr:min. White arrows point to the chromosome arms facing the spindle poles. (B) Western blot to monitor Kif4a levels after RNAi. GAPDH was used as loading control. (C) Chromosome missegregation after Kif4a RNAi (fixed cells). Kinetochores (anti-ACA), α-tubulin, and DNA (DAPI) are indicated. Scale bars, 5 μm. (D and E) Comparison of the frequency of anaphase cells with lagging chromosomes in live (D) and fixed (E) material after Kif4a depletion and/or monastrol washout. (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout in Kif4a-depleted fibroblasts. Dashed bar represents theoretical values for frequencies of lagging chromosomes with small kinetochores if the probability to lag was equal for chromosomes with small or large kinetochores. .
    Figure Legend Snippet: Polar Ejection Forces on Chromosome Arms Ensure Mitotic Fidelity but Are Not Implicated in the Observed Missegregation Bias for Chromosomes with Large Kinetochores (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) in control (top) and Kif4a RNAi (bottom) cells treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Scale bar, 5 μm. Time, hr:min. White arrows point to the chromosome arms facing the spindle poles. (B) Western blot to monitor Kif4a levels after RNAi. GAPDH was used as loading control. (C) Chromosome missegregation after Kif4a RNAi (fixed cells). Kinetochores (anti-ACA), α-tubulin, and DNA (DAPI) are indicated. Scale bars, 5 μm. (D and E) Comparison of the frequency of anaphase cells with lagging chromosomes in live (D) and fixed (E) material after Kif4a depletion and/or monastrol washout. (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout in Kif4a-depleted fibroblasts. Dashed bar represents theoretical values for frequencies of lagging chromosomes with small kinetochores if the probability to lag was equal for chromosomes with small or large kinetochores. .

    Techniques Used: Live Cell Imaging, Stable Transfection, Expressing, Western Blot

    5) Product Images from "Down-regulation of miR-30b-5p protects cardiomyocytes against hypoxia-induced injury by targeting Aven"

    Article Title: Down-regulation of miR-30b-5p protects cardiomyocytes against hypoxia-induced injury by targeting Aven

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-019-0187-4

    Inhibition of miR-30b-5p suppresses hypoxia-induced apoptosis in cardiomyocytes. a Representative captures of flow-cytometric data demonstrating the percent early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+) in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. b Quantitation of A. c Protein expression of Bax and Bcl-2 was detected by western blot analysis in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. GAPDH was used as an internal control. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; *** p
    Figure Legend Snippet: Inhibition of miR-30b-5p suppresses hypoxia-induced apoptosis in cardiomyocytes. a Representative captures of flow-cytometric data demonstrating the percent early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+) in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. b Quantitation of A. c Protein expression of Bax and Bcl-2 was detected by western blot analysis in AC16 cells grown under normoxia or hypoxia with or without miR-30b-5p inhibitor. GAPDH was used as an internal control. *: hypoxia vs. normoxia; #: hypoxia + inhibitor vs. hypoxia + NC; *** p

    Techniques Used: Inhibition, Flow Cytometry, Quantitation Assay, Expressing, Western Blot

    6) Product Images from "MiR-30b-5p regulates the lipid metabolism by targeting PPARGC1A in Huh-7 cell line"

    Article Title: MiR-30b-5p regulates the lipid metabolism by targeting PPARGC1A in Huh-7 cell line

    Journal: Lipids in Health and Disease

    doi: 10.1186/s12944-020-01261-3

    MiR-30b-5p regulates the expression of lipid metabolism-related genes. a protein expression of PPAR-α, SREBP-1, and GLUT1 in miR-30b-5p overexpressed or inhibited Huh-7 cells. b-g the relative quantify of expression of PPAR-α, SREBP-1, and GLUT1 normalized to GAPDH. (I, inhibition; IC, inhibition control; O, overexpression; OC, overexpression control)
    Figure Legend Snippet: MiR-30b-5p regulates the expression of lipid metabolism-related genes. a protein expression of PPAR-α, SREBP-1, and GLUT1 in miR-30b-5p overexpressed or inhibited Huh-7 cells. b-g the relative quantify of expression of PPAR-α, SREBP-1, and GLUT1 normalized to GAPDH. (I, inhibition; IC, inhibition control; O, overexpression; OC, overexpression control)

    Techniques Used: Expressing, Inhibition, Over Expression

    7) Product Images from "Comparative Transcriptome Analysis Reveals Different Host Cell Responses to Acute and Persistent Foot-and-Mouth Disease Virus Infection"

    Article Title: Comparative Transcriptome Analysis Reveals Different Host Cell Responses to Acute and Persistent Foot-and-Mouth Disease Virus Infection

    Journal: Virologica Sinica

    doi: 10.1007/s12250-019-00155-8

    Hspb1 is beneficial for FMDV replication. For simplicity, BHK-21 cells acutely and persistently infected with serotype-O FMDV are abbreviated as BHKOa and BHKOp, respectively. A Protein expression levels of Hspb1 in mock, acutely, and persistently infected BHK-21 cells. B , C BHK-21 cells were transfected with pSUPER-NC or pSUPER-shHspb1 #1, #2, or #3 for 48 h. The mRNA and protein expression levels of Hspb1 were examined by RT-qPCR and Western blot analysis, respectively. D , E BHK-21 cells were transfected with pSUPER-shHspb1 #1 and pSUPER-NC for 24 h and then infected with FMDV at 10 −3 TCID 50 /cell. The mRNA and protein expression levels of 3D and Hspb1 were examined 24 h post infection. F Relative densitometry quantification of 3D protein expression level was quantified by Image Lab software. G BHK-21 cells were transfected with pSUPER-shHspb1 and pSUPER-NC for 24 h and then infected with FMDV at 10 −3 TCID 50 /cell. The relative expression levels of negative stand RNA of 3D were examined 24 h post infection. Relative expression levels were normalized against GAPDH. Three independent replicates were conducted for each sample. Data are expressed as the mean ± SE (n = 3). ** P
    Figure Legend Snippet: Hspb1 is beneficial for FMDV replication. For simplicity, BHK-21 cells acutely and persistently infected with serotype-O FMDV are abbreviated as BHKOa and BHKOp, respectively. A Protein expression levels of Hspb1 in mock, acutely, and persistently infected BHK-21 cells. B , C BHK-21 cells were transfected with pSUPER-NC or pSUPER-shHspb1 #1, #2, or #3 for 48 h. The mRNA and protein expression levels of Hspb1 were examined by RT-qPCR and Western blot analysis, respectively. D , E BHK-21 cells were transfected with pSUPER-shHspb1 #1 and pSUPER-NC for 24 h and then infected with FMDV at 10 −3 TCID 50 /cell. The mRNA and protein expression levels of 3D and Hspb1 were examined 24 h post infection. F Relative densitometry quantification of 3D protein expression level was quantified by Image Lab software. G BHK-21 cells were transfected with pSUPER-shHspb1 and pSUPER-NC for 24 h and then infected with FMDV at 10 −3 TCID 50 /cell. The relative expression levels of negative stand RNA of 3D were examined 24 h post infection. Relative expression levels were normalized against GAPDH. Three independent replicates were conducted for each sample. Data are expressed as the mean ± SE (n = 3). ** P

    Techniques Used: Infection, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Software

    8) Product Images from "MicroRNA deep sequencing in two adult stem cell populations identifies miR-501 as a novel regulator of myosin heavy chain during muscle regeneration"

    Article Title: MicroRNA deep sequencing in two adult stem cell populations identifies miR-501 as a novel regulator of myosin heavy chain during muscle regeneration

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.136051

    Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P
    Figure Legend Snippet: Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P

    Techniques Used: Transfection, RNA Sequencing Assay, Quantitative RT-PCR, Inhibition, Over Expression, Plasmid Preparation, Clone Assay, Mutagenesis, Binding Assay, Construct, Luciferase, Activity Assay, Expressing, FACS, Injection, Derivative Assay

    Inhibition of miR-501 in regenerating skeletal muscle inhibits appearance of myosin heavy chain and formation of large myofibers. (A) Mouse TA muscles were injected with CTX and control antagomir (antCtr) or antagomir-501 (ant501). Antagomir injection was repeated after 3 and 6 days, and muscles were harvested on day 4, 6, 9 or 15. qRT-PCR analysis of miR-501 expression is shown for FACS-isolated MPs or regenerating muscle tissue (CTX TA) at day 4 ( n =4-6, normalized to sno234). (B) Coomassie Blue staining is shown after resolving muscle lysates harvested on day 6 from PBS, antCtr and ant501 treated animals. (C) Western blot analysis for MYH3 and desmin proteins from one TA muscle on day 6 of regeneration ( n =4 mice per group) or for total MYH on day 9 and 15 after CTX injection from both TA muscles ( n =3 mice). Bar graphs show densitometry of western blots normalized to GAPDH or desmin, as indicated. (D) Expression of Myh3 (eMHC) and adult muscle markers myoglobin (Mb) and myostatin transcripts in TA muscle on day 4 and 6 after CTX injection as measured by qRT-PCR. Data are normalized to 18S rRNA and shown relative to day 4 in antCtr group. n =2-3 mice, 4-6 TA muscles per group and time point. (E,F) Frozen muscle sections harvested on day 6 after CTX were probed with MYH3- and laminin-antibodies, and DAPI. Representative pictures are shown in E. Fiber diameter was analyzed based on laminin immunofluorescence and shown relative to the total number of fibers in F. n =7-8 mice per group. All data are presented as mean±s.e.m. * P
    Figure Legend Snippet: Inhibition of miR-501 in regenerating skeletal muscle inhibits appearance of myosin heavy chain and formation of large myofibers. (A) Mouse TA muscles were injected with CTX and control antagomir (antCtr) or antagomir-501 (ant501). Antagomir injection was repeated after 3 and 6 days, and muscles were harvested on day 4, 6, 9 or 15. qRT-PCR analysis of miR-501 expression is shown for FACS-isolated MPs or regenerating muscle tissue (CTX TA) at day 4 ( n =4-6, normalized to sno234). (B) Coomassie Blue staining is shown after resolving muscle lysates harvested on day 6 from PBS, antCtr and ant501 treated animals. (C) Western blot analysis for MYH3 and desmin proteins from one TA muscle on day 6 of regeneration ( n =4 mice per group) or for total MYH on day 9 and 15 after CTX injection from both TA muscles ( n =3 mice). Bar graphs show densitometry of western blots normalized to GAPDH or desmin, as indicated. (D) Expression of Myh3 (eMHC) and adult muscle markers myoglobin (Mb) and myostatin transcripts in TA muscle on day 4 and 6 after CTX injection as measured by qRT-PCR. Data are normalized to 18S rRNA and shown relative to day 4 in antCtr group. n =2-3 mice, 4-6 TA muscles per group and time point. (E,F) Frozen muscle sections harvested on day 6 after CTX were probed with MYH3- and laminin-antibodies, and DAPI. Representative pictures are shown in E. Fiber diameter was analyzed based on laminin immunofluorescence and shown relative to the total number of fibers in F. n =7-8 mice per group. All data are presented as mean±s.e.m. * P

    Techniques Used: Inhibition, Injection, Quantitative RT-PCR, Expressing, FACS, Isolation, Staining, Western Blot, Mouse Assay, Immunofluorescence

    9) Product Images from "Resveratrol alleviates MPTP-induced motor impairments and pathological changes by autophagic degradation of α-synuclein via SIRT1-deacetylated LC3"

    Article Title: Resveratrol alleviates MPTP-induced motor impairments and pathological changes by autophagic degradation of α-synuclein via SIRT1-deacetylated LC3

    Journal: Molecular nutrition & food research

    doi: 10.1002/mnfr.201600111

    RV rescued the loss of nigral TH positive neurons and the decrease of striatal TH protein induced by MPTP Inhibition of SIRT1 with EX527 significantly reversed the effect of RV. (A) Representative microphotographs of dopaminergic neurons stained for TH in the SN. The white arrows indicate the areas showing the most differentiation and higher magnifications of the areas are shown in the rectangle. Scale bars: 50 pm. (B) Statistical results for the relative number of TH-positive neurons in the SN. Values were presented as means ± standard error (n = 4). (C) Western blotting analysis and quantification of relative TH protein abundance in the striatum. (D) Western blotting analysis and quantification of relative caspase 3 and cleaved caspase 3 protein abundance in the striatum. GAPDH protein served as the internal control. Values are presented as means ± standard error (n = 3). **p
    Figure Legend Snippet: RV rescued the loss of nigral TH positive neurons and the decrease of striatal TH protein induced by MPTP Inhibition of SIRT1 with EX527 significantly reversed the effect of RV. (A) Representative microphotographs of dopaminergic neurons stained for TH in the SN. The white arrows indicate the areas showing the most differentiation and higher magnifications of the areas are shown in the rectangle. Scale bars: 50 pm. (B) Statistical results for the relative number of TH-positive neurons in the SN. Values were presented as means ± standard error (n = 4). (C) Western blotting analysis and quantification of relative TH protein abundance in the striatum. (D) Western blotting analysis and quantification of relative caspase 3 and cleaved caspase 3 protein abundance in the striatum. GAPDH protein served as the internal control. Values are presented as means ± standard error (n = 3). **p

    Techniques Used: Inhibition, Staining, Western Blot

    10) Product Images from "A regulatory circuit of circ-MTO1/miR-17/QKI-5 inhibits the proliferation of lung adenocarcinoma"

    Article Title: A regulatory circuit of circ-MTO1/miR-17/QKI-5 inhibits the proliferation of lung adenocarcinoma

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2019.1598762

    Overexpression of miR-17 or silencing of QKI-5 could block the inhibition of Notch pathway caused by circ-MTO1 overexpression in LUAD cells . (A–B) Western blot analysis for the protein expression of NICD, HES1, and Hey2 in control or circ-MTO1-overexpressing A549 (A) and SPC-A1 (B) cells after transfection with miR-17 mimics or QKI-5 si-RNA. GAPDH as an internal reference. (C) Schematic diagram of the mechanism by which the circ-MTO1/miR-17/QKI-5 regulatory loop inhibits lung cancer proliferation by inactivating Notch signaling pathway. ** p
    Figure Legend Snippet: Overexpression of miR-17 or silencing of QKI-5 could block the inhibition of Notch pathway caused by circ-MTO1 overexpression in LUAD cells . (A–B) Western blot analysis for the protein expression of NICD, HES1, and Hey2 in control or circ-MTO1-overexpressing A549 (A) and SPC-A1 (B) cells after transfection with miR-17 mimics or QKI-5 si-RNA. GAPDH as an internal reference. (C) Schematic diagram of the mechanism by which the circ-MTO1/miR-17/QKI-5 regulatory loop inhibits lung cancer proliferation by inactivating Notch signaling pathway. ** p

    Techniques Used: Over Expression, Blocking Assay, Inhibition, Western Blot, Expressing, Transfection

    11) Product Images from "The novel autophagy inhibitor elaiophylin exerts antitumor activity against multiple myeloma with mutant TP53 in part through endoplasmic reticulum stress-induced apoptosis"

    Article Title: The novel autophagy inhibitor elaiophylin exerts antitumor activity against multiple myeloma with mutant TP53 in part through endoplasmic reticulum stress-induced apoptosis

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2017.1345386

    Elaiophylin sequentially induced ER stress. (A) Assessment of 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression using real-time PCR in cells treated with elaiophylin (0.75 μM) for 18 and 36 h and (B) protein expression of GRP78, phospho-PERK (Thr980), ATF4, CHOP and XBP1s detected with western blotting. (C) Western blotting analysis of sequential protein expression changes in GRP78 and MAP1LC3B in cells incubated with elaiophylin (0.75 μM) for successive time points (0, 3, 6, 12, 24 and 36 h). (D) Quantification of the western blots is shown as representative column graphs. GAPDH served as a loading control. The data are shown as the means ± SD (3 independent experiments) and were analyzed using ANOVA (* P
    Figure Legend Snippet: Elaiophylin sequentially induced ER stress. (A) Assessment of 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) expression using real-time PCR in cells treated with elaiophylin (0.75 μM) for 18 and 36 h and (B) protein expression of GRP78, phospho-PERK (Thr980), ATF4, CHOP and XBP1s detected with western blotting. (C) Western blotting analysis of sequential protein expression changes in GRP78 and MAP1LC3B in cells incubated with elaiophylin (0.75 μM) for successive time points (0, 3, 6, 12, 24 and 36 h). (D) Quantification of the western blots is shown as representative column graphs. GAPDH served as a loading control. The data are shown as the means ± SD (3 independent experiments) and were analyzed using ANOVA (* P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation

    12) Product Images from "The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation"

    Article Title: The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa048

    YTHDF1 regulates EIF3C translation in an m 6 A-dependent manner. ( A ) Relative RNA level of EIF3C in A2780 and SKOV3 upon YTHDF1 knockdown. ( B ) Western blot detected the protein level of EIF3C in A2780 and SKOV3 cells upon YTHDF1 knockdown. ( C ) Gene-specific m 6 A qPCR validation of m 6 A levels in A2780 and SKOV3 cells. Primers to m 6 A negative region of EEF1A as the negative control and primers to m 6 A postive region of EEF1A as the positive control. ( D ) YTHDF1 RIP followed by RT-qPCR confirmed the interaction between YTHDF1 and EIF3C mRNA. ( E ) Schematic representation of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. ( F ) RIP-derived RNA and protein in A2780 cells were measured by RT-qPCR and western blot, respectively. GAPDH was used as the negative control in western blot assays. ( G ) Western blot confirmed HA-tagged EIF3C expression in A2780 or SKOV3 cells co-transfected with empty vector, wild-type or mutant Flag-tagged YTHDF1 and wild-type or mutant HA-tagged EIF3C. ( H ) Nascent protein synthesis was detected by HPG incorporation upon YTHDF1 knockdown or EIF3C knockdown in A2780 cells. Scale bar, 100 μm. Data are shown as means ± S.D. *P
    Figure Legend Snippet: YTHDF1 regulates EIF3C translation in an m 6 A-dependent manner. ( A ) Relative RNA level of EIF3C in A2780 and SKOV3 upon YTHDF1 knockdown. ( B ) Western blot detected the protein level of EIF3C in A2780 and SKOV3 cells upon YTHDF1 knockdown. ( C ) Gene-specific m 6 A qPCR validation of m 6 A levels in A2780 and SKOV3 cells. Primers to m 6 A negative region of EEF1A as the negative control and primers to m 6 A postive region of EEF1A as the positive control. ( D ) YTHDF1 RIP followed by RT-qPCR confirmed the interaction between YTHDF1 and EIF3C mRNA. ( E ) Schematic representation of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. ( F ) RIP-derived RNA and protein in A2780 cells were measured by RT-qPCR and western blot, respectively. GAPDH was used as the negative control in western blot assays. ( G ) Western blot confirmed HA-tagged EIF3C expression in A2780 or SKOV3 cells co-transfected with empty vector, wild-type or mutant Flag-tagged YTHDF1 and wild-type or mutant HA-tagged EIF3C. ( H ) Nascent protein synthesis was detected by HPG incorporation upon YTHDF1 knockdown or EIF3C knockdown in A2780 cells. Scale bar, 100 μm. Data are shown as means ± S.D. *P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Quantitative RT-PCR, Mutagenesis, Construct, Derivative Assay, Expressing, Transfection, Plasmid Preparation

    13) Product Images from "The pro-metastasis effect of circANKS1B in breast cancer"

    Article Title: The pro-metastasis effect of circANKS1B in breast cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0914-x

    The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p
    Figure Legend Snippet: The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p

    Techniques Used: Migration, Transfection, Concentration Assay, Multiple Displacement Amplification, Plasmid Preparation, Expressing, Binding Assay

    14) Product Images from "Chromosome Segregation Is Biased by Kinetochore Size"

    Article Title: Chromosome Segregation Is Biased by Kinetochore Size

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.03.023

    Polar Ejection Forces on Chromosome Arms Ensure Mitotic Fidelity but Are Not Implicated in the Observed Missegregation Bias for Chromosomes with Large Kinetochores (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) in control (top) and Kif4a RNAi (bottom) cells treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Scale bar, 5 μm. Time, hr:min. White arrows point to the chromosome arms facing the spindle poles. (B) Western blot to monitor Kif4a levels after RNAi. GAPDH was used as loading control. (C) Chromosome missegregation after Kif4a RNAi (fixed cells). Kinetochores (anti-ACA), α-tubulin, and DNA (DAPI) are indicated. Scale bars, 5 μm. (D and E) Comparison of the frequency of anaphase cells with lagging chromosomes in live (D) and fixed (E) material after Kif4a depletion and/or monastrol washout. (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout in Kif4a-depleted fibroblasts. Dashed bar represents theoretical values for frequencies of lagging chromosomes with small kinetochores if the probability to lag was equal for chromosomes with small or large kinetochores. See also Video S7 .
    Figure Legend Snippet: Polar Ejection Forces on Chromosome Arms Ensure Mitotic Fidelity but Are Not Implicated in the Observed Missegregation Bias for Chromosomes with Large Kinetochores (A) Live-cell imaging of Indian muntjac fibroblasts stably expressing H2B-GFP to visualize the chromosomes (green) in control (top) and Kif4a RNAi (bottom) cells treated with 50 nM SiR-tubulin to label spindle microtubules (magenta). Scale bar, 5 μm. Time, hr:min. White arrows point to the chromosome arms facing the spindle poles. (B) Western blot to monitor Kif4a levels after RNAi. GAPDH was used as loading control. (C) Chromosome missegregation after Kif4a RNAi (fixed cells). Kinetochores (anti-ACA), α-tubulin, and DNA (DAPI) are indicated. Scale bars, 5 μm. (D and E) Comparison of the frequency of anaphase cells with lagging chromosomes in live (D) and fixed (E) material after Kif4a depletion and/or monastrol washout. (F) Frequency of anaphase cells with at least 1 lagging chromosome with small or large kinetochores after monastrol washout in Kif4a-depleted fibroblasts. Dashed bar represents theoretical values for frequencies of lagging chromosomes with small kinetochores if the probability to lag was equal for chromosomes with small or large kinetochores. See also Video S7 .

    Techniques Used: Live Cell Imaging, Stable Transfection, Expressing, Western Blot

    15) Product Images from "APOBEC3A is a prominent cytidine deaminase in breast cancer"

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008545

    APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.
    Figure Legend Snippet: APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

    Techniques Used: Mutagenesis, Expressing, In Vitro, CRISPR, shRNA, Activity Assay, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot

    16) Product Images from "Hepatoprotective effects of Camellia nitidissima aqueous ethanol extract against CCl4-induced acute liver injury in SD rats related to Nrf2 and NF-κB signalling"

    Article Title: Hepatoprotective effects of Camellia nitidissima aqueous ethanol extract against CCl4-induced acute liver injury in SD rats related to Nrf2 and NF-κB signalling

    Journal: Pharmaceutical Biology

    doi: 10.1080/13880209.2020.1739719

    CNE inhibited the NF-κB signalling pathway in rats with ALI. (A,B) The protein levels of p65, p-p65, IκBα and p-IκBα in liver tissues. Lamin B was used as the control for p65 in nuclear protein, and GAPDH was used as the control for p-p65, IκBα and p-IκBα in cytoplasmic proteins ( n = 3). (C,D) CNE activated the Nrf2 signalling pathway in rats treated with CCl 4 . The protein levels of Nrf2 and HO-1. Lamin B was used as the control for Nrf2 in nuclear protein and GAPDH was used as the control for HO-1 in total proteins. These values were expressed as the ratio of the optical density value of each target protein to that of its control protein. (E) The mRNA levels of Nrf2 and HO-1. The values are expressed as the fold increase of RQ value normalized to the control group values (control = 1; n = 3). The results were represented as mean ± SD.
    Figure Legend Snippet: CNE inhibited the NF-κB signalling pathway in rats with ALI. (A,B) The protein levels of p65, p-p65, IκBα and p-IκBα in liver tissues. Lamin B was used as the control for p65 in nuclear protein, and GAPDH was used as the control for p-p65, IκBα and p-IκBα in cytoplasmic proteins ( n = 3). (C,D) CNE activated the Nrf2 signalling pathway in rats treated with CCl 4 . The protein levels of Nrf2 and HO-1. Lamin B was used as the control for Nrf2 in nuclear protein and GAPDH was used as the control for HO-1 in total proteins. These values were expressed as the ratio of the optical density value of each target protein to that of its control protein. (E) The mRNA levels of Nrf2 and HO-1. The values are expressed as the fold increase of RQ value normalized to the control group values (control = 1; n = 3). The results were represented as mean ± SD.

    Techniques Used:

    17) Product Images from "Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles) Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles"

    Article Title: Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles) Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    doi: 10.1002/jcsm.12560

    TRB3 knockout reduced skeletal muscle interstitial fibrosis in aged mice. Mice were divided into WT young group, WT old group, TRB3 −/− young group, and TRB3 −/− old group. (A) Bright‐field and dark‐field imaging of Sirius Red staining, Masson staining, and immunohistochemical staining of collagen I and collagen III. (B) Collagen volume fraction. (C) Immunohistochemical analyses of collagen I and collagen III expression. (D) Collagen I and collagen III expression detected through western blotting; GAPDH: internal reference. (E) Relative expression of collagen I and collagen III. N = 6–9; * P
    Figure Legend Snippet: TRB3 knockout reduced skeletal muscle interstitial fibrosis in aged mice. Mice were divided into WT young group, WT old group, TRB3 −/− young group, and TRB3 −/− old group. (A) Bright‐field and dark‐field imaging of Sirius Red staining, Masson staining, and immunohistochemical staining of collagen I and collagen III. (B) Collagen volume fraction. (C) Immunohistochemical analyses of collagen I and collagen III expression. (D) Collagen I and collagen III expression detected through western blotting; GAPDH: internal reference. (E) Relative expression of collagen I and collagen III. N = 6–9; * P

    Techniques Used: Knock-Out, Mouse Assay, Imaging, Staining, Immunohistochemistry, Expressing, Western Blot

    Establishment of natural aging animal model. Wild‐type C57 mice were divided into young and old groups. (A) Photograph of mice. (B) Body weight (g). (C) Expression of GLB1, p53, p21, and p16 detected through western blotting; GAPDH: internal reference. (D) Relative protein expression levels of GLB1, p53, p21, and p16. N = 6; ** P
    Figure Legend Snippet: Establishment of natural aging animal model. Wild‐type C57 mice were divided into young and old groups. (A) Photograph of mice. (B) Body weight (g). (C) Expression of GLB1, p53, p21, and p16 detected through western blotting; GAPDH: internal reference. (D) Relative protein expression levels of GLB1, p53, p21, and p16. N = 6; ** P

    Techniques Used: Animal Model, Mouse Assay, Expressing, Western Blot

    18) Product Images from "A novel function of anaphase promoting complex subunit 10 in tumor progression in non-small cell lung cancer"

    Article Title: A novel function of anaphase promoting complex subunit 10 in tumor progression in non-small cell lung cancer

    Journal: Cell Cycle

    doi: 10.1080/15384101.2019.1609830

    APC10 is overexpressed in non-small cell lung cancer and located in the cytoplasm. (a) The protein levels of APC10 were determined by western blot using the paired, tumor-adjacent noncancerous lung tissues (normal, N) and human NSCLC tissues (tumor, T) from 15 NSCLC patients (P1 – P15). (b) The expression of APC10 was quantified in these normal and tumor tissues. The p value was calculated by paired t test. (c) The protein levels of APC10 in two normal lung cell cultures (Beas-2b and HBE) and five NSCLC cell lines (H1299, H358, SPC-A1, A549 and H292) were detected by western blot. (d) The location of APC10 was detected by immunofluorescence staining in five NSCLC cell lines (H1299, H292, A549, H358 and SPC-A1). Cells were stained with anti-APC10 (left panel, red) and DAPI (middle panel, blue). The merged images are shown in the right panel. Scale bar = 50 µm, magnification: 200 × . (e) The nuclear and cytoplasmic fractions were separated and the location of APC10 was detected by western blot in H1299, H292, H358 and SPC-A1 cells. TBP: TATA binding protein, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: APC10 is overexpressed in non-small cell lung cancer and located in the cytoplasm. (a) The protein levels of APC10 were determined by western blot using the paired, tumor-adjacent noncancerous lung tissues (normal, N) and human NSCLC tissues (tumor, T) from 15 NSCLC patients (P1 – P15). (b) The expression of APC10 was quantified in these normal and tumor tissues. The p value was calculated by paired t test. (c) The protein levels of APC10 in two normal lung cell cultures (Beas-2b and HBE) and five NSCLC cell lines (H1299, H358, SPC-A1, A549 and H292) were detected by western blot. (d) The location of APC10 was detected by immunofluorescence staining in five NSCLC cell lines (H1299, H292, A549, H358 and SPC-A1). Cells were stained with anti-APC10 (left panel, red) and DAPI (middle panel, blue). The merged images are shown in the right panel. Scale bar = 50 µm, magnification: 200 × . (e) The nuclear and cytoplasmic fractions were separated and the location of APC10 was detected by western blot in H1299, H292, H358 and SPC-A1 cells. TBP: TATA binding protein, GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Western Blot, Expressing, Immunofluorescence, Staining, Binding Assay

    19) Product Images from "ZYZ-803, a novel hydrogen sulfide-nitric oxide conjugated donor, promotes angiogenesis via cross-talk between STAT3 and CaMKII"

    Article Title: ZYZ-803, a novel hydrogen sulfide-nitric oxide conjugated donor, promotes angiogenesis via cross-talk between STAT3 and CaMKII

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/s41401-019-0255-3

    ZYZ-803 induced the nuclear translocation of STAT3. a HUVECs were treated with ZYZ-803 (1 µM) for 15, 30, 60 min, and then, cytoplasmic and nuclear proteins were isolated for Western blotting. GAPDH and Lamin B1 were used as a loading control for cytoplasmic and nuclear proteins, respectively. b HUVECs were treated with ZYZ-803 (1 µM) for 30 min and then stained with specific antibody (red) or DAPI (blue). Scale bar, 10 μm. n = 3 independent experiments. * P
    Figure Legend Snippet: ZYZ-803 induced the nuclear translocation of STAT3. a HUVECs were treated with ZYZ-803 (1 µM) for 15, 30, 60 min, and then, cytoplasmic and nuclear proteins were isolated for Western blotting. GAPDH and Lamin B1 were used as a loading control for cytoplasmic and nuclear proteins, respectively. b HUVECs were treated with ZYZ-803 (1 µM) for 30 min and then stained with specific antibody (red) or DAPI (blue). Scale bar, 10 μm. n = 3 independent experiments. * P

    Techniques Used: Translocation Assay, Isolation, Western Blot, Staining

    20) Product Images from "Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL"

    Article Title: Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-019-0230-2

    Bcl-2 reduces the caspase-1/-4/-5 generated P30.5 GSDMD fragment but enhances the P10 N-terminal fragment. a Cell lysates from HEK-293T cells transiently expressing the indicated constructs were used for the immunoblots. The Flag-GSDMD-full length, Flag-N-terminal (NT) fragment P30.5, and the Flag-NT fragment P10 proteins were detected with the anti-Flag antibody (upper panels), and the caspase proteins blotted with anti-Myc antibody (middle panels). The expressed Bcl-2 was immunoblotted with anti-Bcl-2 antibody. The GAPDH blot serves as a loading control. b , c BH3-like domain of GSDMD is required for Bcl-2 to regulate GSDMD cleaved by caspase-1, -4 or -5. Immunoblots of cell lysates from HEK-293T cells transiently transfected with Flag-GSDMD or Flag-GSDMD L150Q and the indicated constructs. The full length, P30.5, and P10 N-terminal fragments were detected with anti-Flag antibody (upper panels). Caspase-1/-4/-5 were detected with anti-Myc antibody. Bcl-2 was immunoblotted with anti-Bcl-2 antibody. d Trypan blue exclusion was used to quantitate cell death. The HEK-293T cells were transfected with the indicated constructs for 24 h. Data are means and ± SD of four samples. **p
    Figure Legend Snippet: Bcl-2 reduces the caspase-1/-4/-5 generated P30.5 GSDMD fragment but enhances the P10 N-terminal fragment. a Cell lysates from HEK-293T cells transiently expressing the indicated constructs were used for the immunoblots. The Flag-GSDMD-full length, Flag-N-terminal (NT) fragment P30.5, and the Flag-NT fragment P10 proteins were detected with the anti-Flag antibody (upper panels), and the caspase proteins blotted with anti-Myc antibody (middle panels). The expressed Bcl-2 was immunoblotted with anti-Bcl-2 antibody. The GAPDH blot serves as a loading control. b , c BH3-like domain of GSDMD is required for Bcl-2 to regulate GSDMD cleaved by caspase-1, -4 or -5. Immunoblots of cell lysates from HEK-293T cells transiently transfected with Flag-GSDMD or Flag-GSDMD L150Q and the indicated constructs. The full length, P30.5, and P10 N-terminal fragments were detected with anti-Flag antibody (upper panels). Caspase-1/-4/-5 were detected with anti-Myc antibody. Bcl-2 was immunoblotted with anti-Bcl-2 antibody. d Trypan blue exclusion was used to quantitate cell death. The HEK-293T cells were transfected with the indicated constructs for 24 h. Data are means and ± SD of four samples. **p

    Techniques Used: Generated, Expressing, Construct, Western Blot, Transfection

    Bcl-2 induces caspase-1/-4/-5 to cleave GSDMD at D87. a – c Cell lysates from HEK-293T cells transfected with the Flag-GSDMD, Flag-GSDMD D87A, and Flag-GSDMD D275A along with Myc-Casp-1 ( a ), Myc-Casp-4 ( b ), or Myc-Casp-5 ( c ) in the presence or absence of Bcl-2 were used for immunoblotting. Antibodies raised against amino acids 154–252 of GSDMD were used to detect the C-terminal fragment P42.8 of Flag-GSDMD. The membranes were re-blotted with anti-Flag antibodies to detect the N-terminal fragments of Flag-GSDMD cleaved at D87 or D275. Myc-tagged caspase-1, -4 and -5 were detected with tag specific antibodies. Bcl-2 and GAPDH were detected with specific antibodies. d Bcl-2 recombinant protein reduces the cleavage of GSDMD driven by active caspase-3 in vitro. The immunoprecipitated Flag-GSDMD served as a caspase-3 substrate and was incubated with active caspase-3 with or without Bcl-2 protein in caspase assay buffer. The upper panel shows GSDMD full length, merged IgG heavy chain, IgG light chain and cleaved GSDMD N-terminal P10 at longer exposure (LE). The middle panel shows the shorter exposure (SE). The bottom panel shows the added Bcl-2 recombinant protein.
    Figure Legend Snippet: Bcl-2 induces caspase-1/-4/-5 to cleave GSDMD at D87. a – c Cell lysates from HEK-293T cells transfected with the Flag-GSDMD, Flag-GSDMD D87A, and Flag-GSDMD D275A along with Myc-Casp-1 ( a ), Myc-Casp-4 ( b ), or Myc-Casp-5 ( c ) in the presence or absence of Bcl-2 were used for immunoblotting. Antibodies raised against amino acids 154–252 of GSDMD were used to detect the C-terminal fragment P42.8 of Flag-GSDMD. The membranes were re-blotted with anti-Flag antibodies to detect the N-terminal fragments of Flag-GSDMD cleaved at D87 or D275. Myc-tagged caspase-1, -4 and -5 were detected with tag specific antibodies. Bcl-2 and GAPDH were detected with specific antibodies. d Bcl-2 recombinant protein reduces the cleavage of GSDMD driven by active caspase-3 in vitro. The immunoprecipitated Flag-GSDMD served as a caspase-3 substrate and was incubated with active caspase-3 with or without Bcl-2 protein in caspase assay buffer. The upper panel shows GSDMD full length, merged IgG heavy chain, IgG light chain and cleaved GSDMD N-terminal P10 at longer exposure (LE). The middle panel shows the shorter exposure (SE). The bottom panel shows the added Bcl-2 recombinant protein.

    Techniques Used: Transfection, Recombinant, In Vitro, Immunoprecipitation, Incubation, Caspase Assay

    21) Product Images from "The pro-metastasis effect of circANKS1B in breast cancer"

    Article Title: The pro-metastasis effect of circANKS1B in breast cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0914-x

    The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p
    Figure Legend Snippet: The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p

    Techniques Used: Migration, Transfection, Concentration Assay, Multiple Displacement Amplification, Plasmid Preparation, Expressing, Binding Assay

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    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma
    Article Snippet: .. 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK). .. 2.8 Cell and tissue immunofluorescenceCell or tissue immunofluorescence (IF) staining was performed as previously reported , ; cells were incubated with anti‐αSMA (1:300), FSP1 (1:100), or anti‐CD68 (1:100, ZM‐0464; Zhongshan Golden Bridge Biotechnology Co., Beijing, China) overnight at 4°C.

    Incubation:

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    other:

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    Article Title: S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer
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    Blocking Assay:

    Article Title: MicroRNA-137 suppresses the proliferation, migration and invasion of cholangiocarcinoma cells by targeting WNT2B
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    Proteintech anti gapdh
    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and <t>GAPDH)</t> in exosomes. <t>FASN</t> and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS
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    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS

    Journal: Journal of Ovarian Research

    Article Title: Proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells

    doi: 10.1186/s13048-020-0609-y

    Figure Lengend Snippet: Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS

    Article Snippet: The antibodies used were anti-FASN (ABclonal, A0462), anti-L1CAM (ABclonal, A8555), anti-TSG101 (Proteintech, 14,497–1-AP), anti-GAPDH (Proteintech, 60,004–1-Ig), and anti-β-Actin (Proteintech, 60,008–1-Ig).

    Techniques: Tandem Mass Spectroscopy, Western Blot, Marker, Negative Control, Derivative Assay

    The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: RNA Sequencing Assay, Western Blot, Expressing, Isolation

    ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: Western Blot, Expressing

    Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Cell Culture, Immunofluorescence, Staining, CCK-8 Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Irradiation

    PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey

    Journal: bioRxiv

    Article Title: Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

    doi: 10.1101/2020.07.29.225797

    Figure Lengend Snippet: PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey

    Article Snippet: Antibodies and reagentsAntibodies against the following proteins were used at indicated dilutions: anti-acTub (Sigma-Aldrich T7451, mouse 1:2,000), anti-Arl13b (Proteintech 17711-1-AP, rabbit 1:2,000), anti-IFT88 (Proteintech 13967-1-AP, rabbit 1:200 in IF, 1:1,000 in WB), anti-GFP (raised against 6His-tagged eGFP, rabbit 1:1,000), anti-GPR161 (gift from S. Mukhopadhyay, rabbit 1:500), anti-GLI3 (R & D Systems nachuryAF3690, goat 1:1,000) anti-actin (self-made, rabbit 1:5,000), anti-GAPDH (Proteintech 60004-1-Ig, mouse 1:2,000), anti-PALD1 (Sigma-Aldrich HPA017343, rabbit, IF: 1:250, WB: 1:1,000), anti-SMO (Santa Cruz sc-166685, mouse IgG2a in IF 1:200; Abcam ab236465 for and ), anti-PTCH1 (Abcam ab53715, rabbit, WB 1:1,000), anti-ninein (gift from M. Bornens, rabbit 1:10,000), anti-CEP164 (gift from C. Morison, rabbit, IF 1:2,000), γ-tubulin (Proteintech 66320-1-AP, rabbit 1:1,000).

    Techniques: Activation Assay, SDS Page, Western Blot, Expressing, Immunofluorescence, Microscopy, Fluorescence, Mutagenesis, Modification

    LIF acts via phosphorylation of STAT3 (p-STAT3). (a) Histogram of the relative protein level of p-STAT3/GAPDH shows that the value of P-STAT3/GAPDH was statistically increased in the LIF-treated group ( n = 4), compared with the diabetic control group ( t = 3.85, P = 0.019, n = 4) and the normal control group ( t = −3.20, P = 0.019, n = 4). It is significant among the groups ( F = 6.40, P = 0.018). * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice

    doi: 10.4103/0366-6999.221263

    Figure Lengend Snippet: LIF acts via phosphorylation of STAT3 (p-STAT3). (a) Histogram of the relative protein level of p-STAT3/GAPDH shows that the value of P-STAT3/GAPDH was statistically increased in the LIF-treated group ( n = 4), compared with the diabetic control group ( t = 3.85, P = 0.019, n = 4) and the normal control group ( t = −3.20, P = 0.019, n = 4). It is significant among the groups ( F = 6.40, P = 0.018). * P

    Article Snippet: Blotting were stripped and reprobed with rabbit polyclonal anti-STAT3 (12640S, Cell Signaling Technology, Beverly, MA, USA) and anti-GAPDH antibody (60004-1-Ig, Proteintech, USA) followed by the appropriate secondary antibodies for quantification of the bands.

    Techniques: