Journal: bioRxiv

Article Title: KLF7-Regulated ITGA2 as a Therapeutic Target for Inhibiting Oral Cancer Stem Cells

doi: 10.1101/2024.11.04.621805

Figure Lengend Snippet: A The log2-transformed fold change (FC) in RNA levels between the stem-like cluster with the rest of the clusters and the stem-related pathway with the rest of the pathway. B Immunoblot assay of ITGA2, phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, total-YAP1 protein levels in CAL27 and HSC3 cells after stable silencing ITGA2. C Representative immunofluorescent image showing the expression of YAP1 and DAPI in wt and stable silencing ITGA2 cells. D The sequence and domain in ITGA2 protein. E Co-IP analysis in CAL27 and HSC3 cells transduced with ITGA2 OE and ITGA2 mut plasmid. F Immunoblot assay of phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, total-YAP1 protein levels in CAL27 and HSC3 cells after transduced with ITGA2 OE and ITGA2 mut plasmid. G Representative immunofluorescent image showing the expression of YAP1 and DAPI in CAL27 and HSC3 cells after transduced with ITGA2 OE and ITGA2 mut plasmid.

Article Snippet: After washing and blocking, the primary antibody against YAP1 (66900-1-Ig, Proteintech) was incubated overnight.

Techniques: Transformation Assay, Western Blot, Expressing, Sequencing, Co-Immunoprecipitation Assay, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: KLF7-Regulated ITGA2 as a Therapeutic Target for Inhibiting Oral Cancer Stem Cells

doi: 10.1101/2024.11.04.621805

Figure Lengend Snippet: A The molecular formula of TC-I 15. B Co-IP analysis in CAL27 and HSC3 cells treated with DMSO and TC-I 15. C CAL27 cells were intracardially injected into mice. Seven days later, TC-I 15 was injected (20 mg/kg) into mice via vein every third day for 1 month. D Representative images showing the xenograft model in CA27 cells treated with DMSO or TC-I 15(n = 5 per group) and the growth of tumor grafts was shown. E Immunoblot assay of phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, and total-YAP1 protein levels in xenograft tissue. F Representative FACS plots and quantification of CD133+cells in the xenograft tissues. G CAL27 cells were intracardially injected into mice. Seven days later, TC-I 15 (20 mg/kg) and plastin(5 mg/kg) were injected into nude mice via vein every third day for 1 month. H Representative images showing the xenograft model in CA27 cells treated with DMSO or TC-I 15(n = 5 per group) and the growth of tumor grafts were shown.

Article Snippet: After washing and blocking, the primary antibody against YAP1 (66900-1-Ig, Proteintech) was incubated overnight.

Techniques: Co-Immunoprecipitation Assay, Injection, Western Blot

Journal: Iranian Journal of Pathology

Article Title: YAP1 and P53 Expression in Papillary Thyroid Carcinoma

doi: 10.30699/IJP.2023.553716.2897

Figure Lengend Snippet: Immunohistochemical expression of YAP1 in PTC; (A) positive cytoplasmic staining X10, (B)negative staining X40

Article Snippet: The slices were incubated with primary antibodies against YAP1 (1:200, clone (G-6), Santa Cruz Biotechnology, Dallas, Texas, USA) and p53 (1:1000, clone DO-7; Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Immunohistochemical staining, Expressing, Staining, Negative Staining

Journal: Iranian Journal of Pathology

Article Title: YAP1 and P53 Expression in Papillary Thyroid Carcinoma

doi: 10.30699/IJP.2023.553716.2897

Figure Lengend Snippet: PTC patients' Characteristics According to the immunohistochemical expression of YAP1

Article Snippet: The slices were incubated with primary antibodies against YAP1 (1:200, clone (G-6), Santa Cruz Biotechnology, Dallas, Texas, USA) and p53 (1:1000, clone DO-7; Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Cancer Biology & Therapy

Article Title: miR-506 is a YAP1-dependent tumor suppressor in laryngeal squamous cell carcinoma

doi: 10.1080/15384047.2018.1564569

Figure Lengend Snippet: Categorization of LSCC patients and statistical analysis of their correlations with the expression levels of miR-506 and YAP1.

Article Snippet: The primary antibodies against YAP1 and caspase-3 were obtained from Cell Signaling Technology, Inc..

Techniques: Expressing

Journal: Cancer Biology & Therapy

Article Title: miR-506 is a YAP1-dependent tumor suppressor in laryngeal squamous cell carcinoma

doi: 10.1080/15384047.2018.1564569

Figure Lengend Snippet: Correlations between miR-506 and Yap1 in LSCC patients.

Article Snippet: The primary antibodies against YAP1 and caspase-3 were obtained from Cell Signaling Technology, Inc..

Techniques: Expressing

Journal: Scientific Reports

Article Title: Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

doi: 10.1038/s41598-022-13777-y

Figure Lengend Snippet: Establishment and characterization of Bactrian camel and llama AEC cultures. ( A ) Immunofluorescence analysis showing the development of tight-junctions (ZO-1, white) and ciliogenesis (β-tubulin, red) in Bactrian camel and llama AEC cultures over time from 1-day to 4 weeks post ALI exposure. The cells were counterstained with DAPI (blue) to visualize the nuclei. ( B,C ) Ciliogenesis quantification of camel and llama AEC cultures overtime, respectively. Ciliation was quantified by measuring the area above a fluorescence intensity threshold of five random images acquired per condition. ( D ) Transepithelial electrical resistance (TEER) measurement of camel and llama AEC cultures overtime during the differentiation. ( E ) Epithelial morphology of ex vivo tissues (upper panel) and well-differentiated camel and llama AEC cultures (lower panel). ( F ) DPP4 expression in well-differentiated camel and llama AEC cultures, with Vero cells as a positive control. Scale bar is 20 µm.

Article Snippet: Alexa-Fluor ® 647-labelled rabbit anti β-tubulin IV (Cell Signalling Technology, 9F3) and Alexa-Fluor ® 594-labelled mouse antibody against ZO-1 (Thermo Fisher Scientific, 1A12) were used to visualize cilia and tight junctions, respectively.

Techniques: Immunofluorescence, Fluorescence, Ex Vivo, Expressing, Positive Control

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21 +/+ and Trim21 −/− mice. b Volcano plots of DEPs in BMSCs from Trim21 +/+ and Trim21 −/− mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc-VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1 + OBs derived from Trim21 +/+ and Trim21 −/ − mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Next, 1% goat serum was used to block tissues at 37 °C for 30 min. For IHC staining, sections were treated with primary antibody against YAP1 (CST, Cat# 14074, USA, 1:200) overnight at 4 °C.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. c , d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g , h Representative images of histological sections of the tibia that were stained with TRAP in Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Next, 1% goat serum was used to block tissues at 37 °C for 30 min. For IHC staining, sections were treated with primary antibody against YAP1 (CST, Cat# 14074, USA, 1:200) overnight at 4 °C.

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk . Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix . In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: Next, 1% goat serum was used to block tissues at 37 °C for 30 min. For IHC staining, sections were treated with primary antibody against YAP1 (CST, Cat# 14074, USA, 1:200) overnight at 4 °C.

Techniques: Expressing, Activation Assay

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Primer sequences used in reverse transcription-polymerase chain reaction analysis.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Knockdown of YAP1 and mTOR inhibited the progression of bladder cancer. (A and B) RT-PCR and western blot assays were performed to test the expression of YAP1 and mTOR in bladder cancer and normal bladder tissues ( *** P<0.001). (C) Immunohistochemistry was used to evaluate the expression patterns of the YAP1 and mTOR proteins in three matched pairs of bladder cancer and normal bladder tissues (magnification, x200). (D and E) Immunohistochemistry scores of mTOR and YAP1 staining in 20 paired bladder cancer and adjacent normal tissues. (F and G) RT-PCR was used to analyze the knockdown efficien-cies of si-YAP1 and si-mTOR ( * P<0.05, ** P<0.01). (H and I) HT-1376 and J82 cells were transfected with si-NC, si-YAP1, si-mTOR or si-YAP1 + si-mTOR for 0, 24, 48, 72 and 96 h; then, CCK-8 assay was performed to evaluate cell proliferation. (J) HT-1376 and J82 cells were transfected with si-NC, si-YAP1, si-mTOR or si-YAP1 + si-mTOR for 48 h; then, cells were collected for flow cytometry assay to detect cell apoptosis. (K) Western blotting was performed to detect the expression of apoptosis-related proteins, such as caspase 3/9 and cleaved-caspase3/9 after 48 h of cell transfection (H-K, vs. si-NC group, * P<0.05; vs. si-YAP1 group, # P<0.05; NC, negative control). YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining, Transfection, CCK-8 Assay, Flow Cytometry, Negative Control, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Crosstalk between the YAP1 and mTOR proteins. (A and B) HT-1376 and J82 cells were transfected with OE-mTOR and OE-NC; then cells were harvested and subjected to RT-PCR and western blot assays to determine the expression of mTOR at the mRNA and protein levels, respectively. (C-F) RT-PCR and western blot assays were performed to determine the mRNA and protein expression of YAP1 and TEAD after 48 h of HT-1376 and J82 cell transfection with OE-mTOR or OE-NC. (G) Immunofluorescence assay was performed to evaluate the effects of subcellular location of the YAP1 protein. (H) Statistical analysis of the fluorescence intensity of the YAP1 protein ( * P<0.05, ** P<0.01; NC, negative control). YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; RT-PCR, reverse transcription-polymerase chain reaction; TEAD, TEA domain transcription factor.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Fluorescence, Negative Control, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Upregulation of YAP1 promoted the expression and protein stability of mTOR. (A and B) HT-1376 and J82 cells were transfected with OE-YAP1 and OE-NC; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the expression of YAP1. (C and D) western blotting assays were performed to determine the protein expression and phosphorylation of mTOR, p-mTOR, p-eIF4E, eIF4E, p-rpS6 and rpS6 after HT-1376 and J82 cells were transfected with OE-YAP1 or OE-NC. (E and F) After HT-1376 and J82 cells were transfected with OE-YAP1 and OE-NC for 24 h, they were incubated with 100 µg/ml CHX for 0, 1, 2, 4, 8 and 24 h; then, cells were harvested and protein samples were extracted for western blotting with mTOR antibody. (G) Immunoprecipitation (IP) assay was performed to explore the effects of YAP1 upregulation or MG132 on mTOR expression and ubiquitination in HT-1376 and J82 cells ( * P<0.05, ** P<0.01). YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; RT-PCR, reverse transcription-polymerase chain reaction; eIF, eukaryotic translation initiation factor; rpS6, ribosomal protein s6; CHX, cycloheximide.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Incubation, Immunoprecipitation, Ubiquitin Proteomics, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Interaction between YAP1 protein and mTOR protein in HT-1376 and J82 cells. (A) Immunofluorescence assay was performed to evaluate the subcellular localization of the YAP1 and SKP2 proteins. (B) Duolink assay was performed to evaluate the subcellular localization of the YAP1 and mTOR proteins. (C and D) Western blotting was performed to assess the expression of flag and YAP1 after J82 and HT-1376 cells were transfected with flag-tag or YAP1-flag-tag vector ( * P<0.05). (E) Immunoprecipitation assay was used to assess the combination of YAP1 and mTOR proteins ['input' refers to total protein lysate and 'eluent' refers to the immune complex pulled down by YAP1 antibody or flag antibody; beads were used as a negative control (NC)]. YAP1, Yes-associated protein 1; mTOR, mammalian target of rapamycin; SKP2, S-phase kinase-associated protein 2.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Immunofluorescence, Western Blot, Expressing, Transfection, FLAG-tag, Plasmid Preparation, Immunoprecipitation, Negative Control

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Detection of the interaction between YAP1 and SKP2. (A and B) HT-1376 and J82 cells were transfected with siRNAs-YAP1; then, cells were harvested and subjected to RT-PCR and western blot assays to determine the knockdown efficiency ( ** P<0.01, *** P<0.001). After (C and D) HT-1376 and (E and F) J82 cells were transfected with si-YAP1, si-NC, OE-YAP1 and OE-NC, RT-PCR and western blot assays were performed to determine the mRNA and protein levels of CDC4, SKP2, RCHY1, MDM2, UBE3A and SMURF1 (si-YAP1 vs. si-NC group, * P<0.05, ** P<0.01; OE-YAP1 vs. OE-NC group, # P<0.05, ## P<0.01, ### P<0.001). (G and H) Immunofluorescence and Duolink assays were performed to evaluate the subcellular localization of the YAP1 and SKP2 proteins. (I) Immunoprecipitation assay was used to assess the combination between YAP1 and SKP2 proteins in HT-1376 and J82 cells ['input' refers to total protein lysate and 'eluent' refers to the immune complex pulled down by YAP1 antibody; beads were used as a negative control (NC)]. YAP1, Yes-associated protein 1; SKP2, S-phase kinase-associated protein 2; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Immunofluorescence, Immunoprecipitation, Negative Control, Reverse Transcription, Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Interaction of YAP1 and mTOR promotes bladder cancer progression

doi: 10.3892/ijo.2019.4922

Figure Lengend Snippet: Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (A and B) HT-1376 and J82 cells were trans-fected with siRNAs-SKP2; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the knockdown efficiency of SKP2 ( ** P<0.01, *** P<0.001). (C) Immunoprecipitation assay was used to assess the effects of the YAP1/SKP2 axis on mTOR protein expression and ubiquitination. (D and E) Western blotting was performed to test the expression and phosphorylation of mTOR, p-mTOR, p-eIF4E, elF4E, p-rpS6 and rpS6 following cell transfection. (F and G) The effects of the YAP1/SKP2 axis on cell proliferation were determined by CCK-8 analysis. Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (H) The effects of the YAP1/SKP2 axis on cell apoptosis were assessed by flow cytometry analysis. (I and J) Western blotting was performed to detect the expression of apoptosis-related proteins, such as caspase 3/9 and cleaved-caspase3/9 after 48 h of cell transfection (D-J, OE-YAP1 + si-NC group or OE-NC + si-SKP2 group vs. OE-NC + si-NC group, * P<0.05, ** P<0.01, *** P<0.001; OE-YAP1 + si-SKP2 group vs. OE-YAP1 + si-NC group, # P<0.05, ## P<0.01; NC, negative control). YAP1, Yes-associated protein 1; SKP2, S-phase kinase-associated protein 2; RT-PCR, reverse transcription-polymerase chain reaction; eIF, eukaryotic translation initiation factor; rpS6, ribosomal protein s6.

Article Snippet: Then, cell lysate containing 200 μg protein was incubated with Dynabeads ® Protein G (Thermo Fisher Scientific, Inc.) for 1 h, and incubated with 2 μg antibody against YAP1 (cat. no. PA5-78321, Invitrogen; Thermo Fisher Scientific, Inc.), mTOR (cat. no. PA5-34663, Invitrogen; Thermo Fisher Scientific, Inc.) flag (cat. no. 8146, Cell Signaling Technology, Inc.), or beads (negative control) overnight at 4°C, followed by incubation with Dynabeads ® Protein G for another 1 h to form the immune complex, which was considered as the 'Elute' sample.

Techniques: Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Immunoprecipitation, Expressing, Phospho-proteomics, Transfection, CCK-8 Assay, Flow Cytometry, Negative Control, Reverse Transcription, Polymerase Chain Reaction