Journal: Frontiers in oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2.

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: FIGURE 5 | RANBP2 is the key target of YTHDF1 in cervical cancer. (A) Western blot detecting the protein level of RANBP2 in Hela and Siha cells upon YTHDF1 knockdown. (B) RT-qPCR detecting relative RNA level of RANBP2 in Hela and Siha upon YTHDF1 knockdown. (C) RIP-PCR assays detecting the interactions between YTHDF1 and RANBP2 mRNA in Siha cells. IgG was used as an internal control. GAPDH was used as the negative control in western blot assays. (D) meRIP-PCR assays detecting the m6A modification of RANBP2 mRNA in Siha cells. (E) Schematic of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. (F) RIP-derived RNA and protein of wild-type (YTHDF1-wt) group and mutant (YTHDF1-mut) group in Hela cells were measured by RT-qPCR and western blot after immunoprecipitation by using the antibody specific to Flag, respectively. GAPDH was used as the negative control in western blot assays. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The antibodies used for western blot are as follows: YTHDF1 (ProteinTech, 1:1000), RANBP2 (ProteinTech, 1:1000), GAPDH (ZSGB-BIO, 1:2000), Flag-tag (MBL, 1:5000).

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, Control, Negative Control, Mutagenesis, Construct, Derivative Assay, Immunoprecipitation

Journal: Frontiers in oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2.

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: FIGURE 6 | RANBP2 plays an oncogenic role in cervical cancer cells. (A) Detection of RANBP2 knockdown in Hela and Siha cell lines by western blot. (B) The effect of RANBP2 knockdown on cell growth was determined by CCK-8 assays. (C) Colony formation assays were performed in RANBP2 knockdown and control cells. (D, E) Migration and invasion assays of Hela and Siha cells upon RANBP2 knockdown. Scale bar, 200 mm. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The antibodies used for western blot are as follows: YTHDF1 (ProteinTech, 1:1000), RANBP2 (ProteinTech, 1:1000), GAPDH (ZSGB-BIO, 1:2000), Flag-tag (MBL, 1:5000).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Control, Migration

Journal: Frontiers in oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2.

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: FIGURE 7 | Knockdown of RANBP2 suppressed the proliferation, migration and invasion of YTHDF1-overexpressing Hela and Siha cells. (A) Colony formation assays were performed in YTHDF1-overexpressing Hela and Siha cells infected with the RANBP2 shRNA or controls. (B) The proliferation ability of YTHDF1- overexpressing Hela and Siha cells upon RANBP2 knockdown was assessed by CCK-8 assays. (C, D) Migration and invasion YTHDF1-overexpressing Hela (C) and Siha (D) cells upon RANBP2 knockdown was detected by transwell assays. Scale bar, 200 mm. *P < 0.05,**P < 0.01, ***P < 0.001.

Article Snippet: The antibodies used for western blot are as follows: YTHDF1 (ProteinTech, 1:1000), RANBP2 (ProteinTech, 1:1000), GAPDH (ZSGB-BIO, 1:2000), Flag-tag (MBL, 1:5000).

Techniques: Knockdown, Migration, Infection, shRNA, CCK-8 Assay

Journal: Frontiers in oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2.

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: FIGURE 8 | The expression of RANBP2 is positively correlated with YTHDF1 in cervical cancer. (A) Representative immunohistochemical images of RANBP2 protein expression in cervical cancer tissues and cervical epithelium tissues. Scale bar, 100 mm. (B) The quantitative analysis of RANBP2 expression in cervical cancer tissues and cervical epithelium tissues assessed by immunohistochemistry. (C) Spearman’s correlation analysis of RANBP2 and YTHDF1 expression in cervical cancer tissues. (D) Representative immunohistochemical images of YTHDF1 and RANBP2 in the cervical cancer tissues. Scale bar, 100 mm. Data are shown as means ± S.D. *P < 0.05.

Article Snippet: The antibodies used for western blot are as follows: YTHDF1 (ProteinTech, 1:1000), RANBP2 (ProteinTech, 1:1000), GAPDH (ZSGB-BIO, 1:2000), Flag-tag (MBL, 1:5000).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: PLoS neglected tropical diseases

Article Title: A novel family of cyst proteins with epidermal growth factor repeats in Giardia lamblia.

doi: 10.1371/journal.pntd.0000677

Figure Lengend Snippet: Figure 3. Analysis of egfcp1 gene expression. (A) RT-PCR and quantitative real time PCR analysis of egfcp1 gene expression. RNA samples were prepared from G. lamblia wild-type non-transfected WB cells cultured in growth (Veg) or encystation medium and harvested at 24 h (Enc). RT-PCR and real time PCR were preformed using primers specific for egfcp1, cwp1, ran, and glycyl t-RNA synthetase genes. Representative results are shown on the left panel. Transcript levels of specific genes were normalized to glycyl t-RNA synthetase (control) transcript levels [50]. Fold changes in mRNA expression are shown as the ratio of transcript levels in encysting cells relative to vegetative cells. Results are expressed as the means 6 S.E. of at least three separate experiments (right panel). (B) EGFCP1 protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg) or encystation medium (Enc) for 2, 5, 9, and 24 h and then subjected to SDS-PAGE and Western blot (left). The blot was probed by anti- EGFCP1 antibody. Representative results are shown. The results from Western blot analysis of nonreduced proteins are shown at right panel. Equal amounts of proteins loaded were confirmed by SDS-PAGE and Coomassie blue staining (lower panels). (C) Localization of EGFCP1. The wild-type non- transfected WB cells were cultured in growth (Veg) or encystation medium (Enc) and harvested at 2 h or 24 h, and then subjected to immunofluorescence analysis using anti-EGFCP1 antibody (1/300) for detection (upper panels). The lower panels show the DAPI staining of cell nuclei. EGFCP1 was localized to the ER in a vegetative trophozoite (Veg). During encystation, EGFCP1 was localized to the ER and some big vesicles in a 2 h encysting trophozoite (2 h Enc) and to the ESVs in a 24 h encysting trophozoite (24 h Enc). In the cyst stage, EGFCP1 was localized to the cyst wall and weakly to cell body (Cyst). Most (.80%) cells or cysts are positive stained. (D) The pNEGFCP1 and pPTEGFCP1 plasmids. A neo or pac gene is under the control of the 59- and 39-flanking regions of the ran (dotted box) or gdh (slashed box) gene. The egfcp1 gene is under the control of its own 59-flanking region (open boxes) or constitutively expressed a2-tubulin promoter (striated box) and the 39-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the AU1 (for pNEGFCP1 plasmid) or HA (for pPTEGFCP1 plasmid) epitope tag. The filled gray box indicates the coding sequence of the signal peptide. The arrows show the directions of gene transcription. (E) EGFCP1 protein levels in pNEGFCP1 stable transfectants. The pNEGFCP1 stable transfectants were cultured in growth medium (Veg) or encystation medium and harvested at 24 h (Enc). AU1-tagged EGFCP1 protein was detected using an anti-AU1 antibody by Western blot analysis of reduced proteins. Coomassie-stained total protein loading control is shown below. (F) EGFCP1 protein levels in pPTEGFCP1 stable transfectants. The pPTEGFCP1 stable transfectants were cultured in growth medium (Veg) or encystation medium and harvested at 24 h (Enc). HA-tagged EGFCP1 protein was detected using an anti-HA antibody by Western blot analysis of reduced proteins. Coomassie-stained total protein loading control is shown below. doi:10.1371/journal.pntd.0000677.g003

Article Snippet: Western blots were also probed with anti-EGFCP1 antibody (1/10000), anti-CWP1 antibody (1/10000) [30] or anti-human RAN antibody (1/5000) (Santa Cruz Biotechnology), and detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (1/ 5000; Pierce) and enhanced chemiluminescence (GE Healthcare).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, Cell Culture, Control, Expressing, SDS Page, Western Blot, Staining, Immunofluorescence, Sequencing, Plasmid Preparation

Journal: PLoS neglected tropical diseases

Article Title: A novel family of cyst proteins with epidermal growth factor repeats in Giardia lamblia.

doi: 10.1371/journal.pntd.0000677

Figure Lengend Snippet: Figure 5. Overexpression of EGFCP1 increased the levels of cyst formation. (A) Diagrams of the 59n5N-Pac, ran32, and pPTEGFCP1nsp plasmid. The expression cassettes of the pac and egfcp1 genes (open box) are the same as in Fig. 3D. The ran32 plasmid constains a luciferase gene under the control of the 32 bp ran promoter (dotted box). The pPTEGFCP1nsp plasmid contains an egfcp1 gene lacking the coding sequence for the predicted signal peptide sequence (nucleotides 4–48) (gray box in Fig. 3D). (B) Localization of an EGFCP1 mutant with a deletion of signal peptide (EGFCP1nsp). The pPTEGFCP1nsp stable transfectants were cultured in encystation medium and harvested at 24 h, and then subjected to immunofluorescence analysis using anti-HA antibody for detection (left panel). The right panel shows the DAPI staining of cell nuclei. (C) Cyst count. The 59n5N-Pac, pPTEGFCP1 and pPTEGFCP1nsp stable transfectants were cultured in encystation medium for 24 h, treated with water for 5 times, and subjected to cyst count. The sum of total cysts is expressed as relative expression level over control. Values are shown as means 6 S.E. (D) Deletion of signal peptide reduced the levels of the EGFCP1 protein. The 59n5N-Pac, pPTEGFCP1, and pPTEGFCP1nsp stable transfectants were cultured in encystation medium for 24 h and then subjected to SDS-PAGE and Western blots. The blots were probed by anti-HA, anti-EGFCP1, anti- CWP1, and anti-RAN antibodies. Equal amounts of proteins loaded were confirmed by detection of giardial RAN protein and Coomassie-stained total protein loading control (in the bottom panel). Representative results are shown. (E) Deletion of signal peptide reduced the levels of the egfcp1 transcripts. The 59n5N-Pac, pPTEGFCP1, and pPTEGFCP1nsp stable transfectants were cultured in encystation medium for 24 h and then subjected to RT-PCR (left panel) and quantitative real time PCR analysis (right panel). RT-PCR was preformed using primers specific for total egfcp1, exogenous egfcp1-ha, endogenous egfcp1, cwp1, ran, and glycyl t-RNA synthetase genes. Real time PCR was preformed using primers specific for total egfcp1, exogenous egfcp1-ha, endogenous egfcp1, cwp1, ran and glycyl t-RNA synthetase genes. Transcript levels of specific genes were normalized to glycyl t-RNA synthetase (control) transcript levels [50]. Fold changes in mRNA expression are shown as the ratio of transcript levels in pPTEGFCP1 or pPTEGFCP1nsp cell line relative to the 59n5N-Pac cell lines. Results are expressed as the means 6 S.E. of at least three separate experiments. N.D. = Not determined. (F) Release of EGFCP1 into medium during encystation. 59D5N-Pac, pPTEGFCP1, and pPTEGFCP1nsp stable transfectants were cultured in vegetative growth medium (Veg) or encystation medium (Enc) for 24 h. The cultured media were immunoprecipitated with the anti-HA antibody and then analyzed by Western blot using anti-HA antibody. doi:10.1371/journal.pntd.0000677.g005

Article Snippet: Western blots were also probed with anti-EGFCP1 antibody (1/10000), anti-CWP1 antibody (1/10000) [30] or anti-human RAN antibody (1/5000) (Santa Cruz Biotechnology), and detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (1/ 5000; Pierce) and enhanced chemiluminescence (GE Healthcare).

Techniques: Over Expression, Plasmid Preparation, Expressing, Luciferase, Control, Sequencing, Mutagenesis, Cell Culture, Immunofluorescence, Staining, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: PLoS ONE

Article Title: An Interaction Network Predicted from Public Data as a Discovery Tool: Application to the Hsp90 Molecular Chaperone Machine

doi: 10.1371/journal.pone.0026044

Figure Lengend Snippet: (A) PPI map of the proteins (red nodes) in the GO module “nucleocytoplasmic transport” of the Aha1-Hsp90 PPI subset of . It contains potential and known (dashed and full line edges, respectively) Aha1 interactions. The Hsp90 client protein GR was also integrated into the predicted PPI (upper panel). The co-immunoprecipitation assays of panel B allowed the experimental confirmation and new demonstration of PPIs as indicated by red and green edges, respectively (lower panel). (B) Co-immunoprecipitation experiment demonstrating interactions between Aha1 or exportin-1 (XPO1) and components of the GO module “nucleocytoplasmic transport” shown in panel A. Flag-tagged Aha1, exportin-1, and GPR30 as an unrelated control protein were exogenously expressed in 293T cells. IPO4, importin-4; KPNA5, importin-α6. (C) and (D) Nuclear localization of GR in mouse fibroblasts with and without Aha1. Panel C shows representative micrographs of the localization of Tom.GR with our without treatment with dexamethasone (Dex) for 40 min, and an immunoblot on the right verifying the absence of Aha1 in the Aha1-null fibroblasts. Panel D shows the nuclear accumulation of Tom.GR over time in wild-type (▴) and Aha1-null (▾) cells. Nuclear localization of GR was initiated at time zero with the addition of 10 nM dexamethasone. Data points are the means with standard errors of three independent experiments where ∼100 cells were counted for each time point. *, significantly different with p<0.005.

Article Snippet: The rabbit polyclonal serum against Hsp90α (PA3-013) was from Affinity BioReagents (Golden, CO, USA); mouse monoclonal H90-10 against Hsp90β was kindly provided by Prof. David O. Toft (Mayo Clinic, Rochester, USA); rabbit polyclonal sera against KPNA5 and IPO4 were from ProteinTech Group (Chicago, USA); the mouse monoclonal M2 against the Flag epitope was from Sigma Chemical Co. (St. Louis, MO, USA).

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Journal of Translational Medicine

Article Title: Targeting deubiquitinase USP7-mediated stabilization of XPO1 contributes to the anti-myeloma effects of selinexor

doi: 10.1186/s12967-025-06068-3

Figure Lengend Snippet: The degradation of XPO1 protein exhibits a positive correlation with the sensitivity of MM cells to SEL treatment. A , B and C Cell viability of MM cell lines treated with increasing concentrations of drugs for 48 h, and the IC50 of SEL ( A ), ELT ( B ), VER ( C ) for each cell line. D Cell apoptosis rates of different MM cell lines after treated with indicated concentrations of SEL for 48 h. E Representative immunoblotting images and quantitative analysis of XPO1 protein levels in different MM cell lines treated with increasing concentrations of SEL for 24 h. F and G Immunofluorescent staining of RanBP1(green) in NCI-H929 ( F ) and MM.1S ( G ) cells after treated with DMSO, MG132 (10 μM) and /or SEL (500 nM) for 12 h. Nucleus were stained with DAPI (blue). The data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Antibodies against the following proteins were purchased from Proteintech Technology: RanBP1 (27,804–1-AP), α-Tubulin (66,031–1-Ig), β-actin (66,009–1-Ig).

Techniques: Western Blot, Staining

Journal: bioRxiv

Article Title: An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

doi: 10.1101/588798

Figure Lengend Snippet: Validation of the changes in protein level observed by mass spectrometry between negative control, DDX21 knock-down and WT or M4-DDX21 recovery samples by Western blot. DDX21 knock-down and recovery (top) affects the signal from the MAGED2 antibody, but not the antibodies for CNOT3, XPO6 or Tubulin.

Article Snippet: The following antibodies were used: DDX21 (Novus NB100-1716), MAGED2 (ProteinTech 15252-1-AP), TRAIL-R2 (AbCam EPR19310, AB199357), CNOT3 (ProteinTech 11135-1-AP), XPO6 (ProteinTech 11408-1-AP), gapdh, tubulin, goat anti rabbit secondary, goat anti mouse secondary.

Techniques: Mass Spectrometry, Negative Control, Western Blot

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

doi: 10.1016/j.bbadis.2019.01.011

Figure Lengend Snippet: TGF-β1 signaling in mouse cardiac tissue extracts and cardiac fibroblasts. A. Immunoblot analysis of p-Smad2 and p-Smad3 in cardiac tissue extracts from WT and Mcpt4−/− mice at 28 days post-MI. B. Immunoblot analysis of p-Smad2, p-Smad3, and α-SMA in fibroblasts from WT and Mcpt4−/− mice with and without TGF-β stimulation for 30 min (for p-Smad2 and p-Smad3) or 36 hours (for α-SMA). C. Immunoblot analysis of p-Smad2 and p-Smad3 in cytosolic and nuclear fractions of fibroblasts from WT and Mcpt4−/− mice. The same blot was reprobed for GAPDH and histone H3 to ensure cytosol and nucleus separation. D. Immunoblot analysis of nuclear membrane importer importin-β and exporter RanBP3 in fibroblasts from WT and Mcpt4−/− mice. The same blots were reprobed for GAPDH to examine equal protein loading in panels A, B, and D. Data are representative of three independent experiments.

Article Snippet: For immunoblot analysis, an equal amount of protein lysate from each cell type preparation or myocardium tissue extract was separated by SDS-PAGE, blotted, and detected with different antibodies, including cathepsin K (CatK) (1:1000, Cat# PB9856, BOSTER, Pleasanton, CA), cathepsin S (CatS) (1:1000) [ 32 ], cathepsin B (CatB) (1:1000, Cat# PC41–100UG, Sigma-Aldrich), cathepsin L (CatL) (1:1000, Cat# 168–10557, RayBiotech, Inc., Norcross, GA), mMCP4 (1:1000), α-SMA (1:1000, Cat# 14968s, Cell Signaling Technology), GAPDH (1:1000, Cat# 2118S, Cell Signaling Technology), p-Smad2 (1:1000, Cat# 3101s, Cell Signaling Technology), p-Smad3 (1:1000, Cat# 9520s, Cell Signaling Technology), importin-β (1:1000, Cat# 8673S, Cell Signaling Technology) and RanBP3 (1:1000, Cat# SC-373678, Santa Cruz Biotechnology, Dallas, TX), α-actin (1:1000, Cat# A5441-.2ML, Sigma-Aldrich), and histone-H3 (1:1000, Cat# ab1791, Abcam).

Techniques: Western Blot, Membrane