Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 2. RT-PCR of 5*and 3* domains of ACT cDNA from hip- pocampus of AD and normal brains. A, RT-PCR of the 59 domain of ACT cDNA, detected by DNA agarose gels. RT-PCR with primers 1 and 2 generated a 790-bp DNA band from poly(A)1 RNA isolated from hippocampus (H) of AD and normal (N) brains (lanes 1 and 2, respec- tively) as well as from human liver (L) (lane 3). B, RT-PCR of the 39 domain of ACT cDNA. RT-PCR with primers 3 and 4 generated a DNA band of approximately 500 bp from poly(A)1 RNA isolated from hip- pocampus (H) of AD and normal (N) brains (lanes 1 and 2, respectively) as well as from human liver (L) (lane 3).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Reverse Transcription Polymerase Chain Reaction, Generated, Isolation

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 1. Strategy for RT-PCR of ACT cDNA from hippocampus. Primers 1 and 2 and primers 3 and 4 were designed to amplify 59 and 39domains of the hip- pocampus ACT cDNA, respectively. The overlapping 59 and 39 domains were pre- dicted to include the RSL and the NH2 terminus of mature, processed ACT, which begins at the COOH terminus of the signal peptide sequence. Primers 5 and 6 allowed amplification of the 39-UTR of the cDNA. Primer A was used in primer exten- sion analyses of ACT gene transcripts.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 3. Complementary DNA sequence of human hippocampus ACT. The ACT cDNAs obtained from hippocampus of normal brains is illustrated. The open reading frame domains of ACT cDNAs from normal and Alzheimer’s disease brains were identical. The DNA sequence of the 39-UTR domain of the hippocampus ACT cDNA (from normal brain) was also determined. Alignment of the DNA sequences determined for overlapping 59 and 39 PCR fragments indicates the human hippocampus ACT cDNA (shown in bold for nucleotides 111 to 1576), whose deduced primary sequence (shown in bold for residues 27 to 398) corresponds to the mature ACT protein. The positions of primers 1–6 used in RT-PCRs are shown by dotted lines with arrows. Arrows above the His (11) and Asn (13) residues indicate the predicted NH2 terminus of the mature ACT, which lacks the signal sequence. The RSL domain is boxed, with the predicted P1 residue as Leu underlined. Consensus glycosylation sites are indicated by asterisks under the Asn residues as possible sites of glycosylation. The predicted 59-region analyzed by primer extension is shown (not bold) for nucleotides 1–110 (6, 17).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Sequencing, Residue, Glycoproteomics

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 4. Northern blot of ACT mRNA in hippocampus from AD and normal hippocampus as well as liver. Northern blots of total RNA isolated from hippocampus of normal (N) and AD brains (10 mg of RNA each, lanes 1 and 2, respectively) and liver poly(A)1 RNA (1 mg, lane 3) probed with the human ACT cDNA (6), as described under “Experimental Procedures.” Autoradiography of Northern blots (15 h exposure to x-ray film) (lanes 1–3) showed ACT mRNA in hippocampus and a high level of ACT mRNA in liver. Intact ribosomal RNAs were detected by ethidium bromide staining of the RNA samples on dena- turing formaldehyde gels (data not shown). kb, kilobases.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Northern Blot, Isolation, Autoradiography, Staining

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 5. Slot blot of ACT mRNA in hippocampus and liver. Slot blots of poly(A)1 RNA (with the indicated amounts of RNA) from hip- pocampus and liver were performed to compare ACT mRNA levels in these two tissues. Hybridization of slot blots with ACT cDNA as probe was performed identically as described for Northern blots of ACT mRNA (Fig. 4). Northern blots were subjected to autoradiography (15 h exposure to x-ray film) for detection of ACT mRNA.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Dot Blot, Hybridization, Northern Blot, Autoradiography

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 6. Genomic blot of human ACT. DNA agarose (0.8%) gel electrophoresis of KpnI-, HindIII-, or EcoRI- (lanes 1–4 respectively) digested human genomic DNA (10 mg) as well as undigested control DNA (lane 5) was subjected to in situ hybridization with the human liver ACT cDNA as probe (6), as described under “Experimental Proce- dures.” kb, kilobases.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Nucleic Acid Electrophoresis, Control, In Situ Hybridization

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 7. Primer extension of ACT mRNA from hippocampus and liver. Primer extension of poly(A)1 RNA from hippocampus and liver (lanes 1 and 2, respectively) was conducted with 32P-labeled primer 59-CTGCCTCAGGGAGCTGGA-39. The 32P-extended cDNA was ana- lyzed on 8% acrylamide, bis-acrylamide, 7 M urea DNA sequencing gels, with detection of the extended cDNA by autoradiography, as described under “Experimental Procedures.” Arrows indicate the radiolabeled cDNAs of 58 bp obtained by primer extension.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Labeling, DNA Sequencing, Autoradiography

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 8. Deglycosylation of ACT in hippocampus of Alzheimer’s disease and normal brains as well as in liver. Deglycosylation by N-glycosidase F of ACT in tissue extracts from hippocampus of AD and normal brains as well as hu- man liver ACT was assessed by Western blots with anti-ACT serum. Panel A shows ACT in normal (N) and AD hip- pocampus (lanes 1 and 3, respectively). ACT in these tissues was also incubated with (1) N-glycosidase F (lanes 2 and 4, respectively). Panel B shows human liver ACT without and with N-glycosidase F treatment (lanes 1 and 2, respectively).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Western Blot, Incubation

Journal: International journal of molecular sciences

Article Title: Role of Acyl-CoA Thioesterase 7 in Regulating Fatty Acid Metabolism and Its Contribution to the Onset and Progression of Bovine Clinical Mastitis.

doi: 10.3390/ijms252313046

Figure Lengend Snippet: Figure 4. Distribution analysis of ACOT7 in mammary glands of the two groups. (A) Pathological variation in bovine mammary glands of the healthy control group (Con/C; A1) and clinical mastitis groups (CM; A2) based on H&E staining. (B) ACOT7 distribution in bovine mammary glands of the Con/C (B1) and CM (B2) groups based on immunohistochemical staining. (C) Negative control (NC) for Con/C (C1) and CM (C2) groups. (D) Gray values of positive expression of ACOT7 protein quantified using ImageJ 1.44p software. Con/C, control group. CM, clinical mastitis group. NC, negative control; MA: mammary alveoli; MECs, mammary epithelial cells; NEUT, neutrophil. Scale bars, 50 µm (200× magnification). ** represents p < 0.01.

Article Snippet: The slices were incubated with a rabbit anti-ACOT7 primary antibody at a 1:200 dilution (Proteintech, Wuhan, China).

Techniques: Control, Staining, Immunohistochemical staining, Negative Control, Expressing, Software

Journal: International journal of molecular sciences

Article Title: Role of Acyl-CoA Thioesterase 7 in Regulating Fatty Acid Metabolism and Its Contribution to the Onset and Progression of Bovine Clinical Mastitis.

doi: 10.3390/ijms252313046

Figure Lengend Snippet: Figure 5. Co-localization analysis of ACOT7 in mammary glands of the two groups. (A–D) Co- localization analysis of ACOT7 in bovine mammary glands of the Con/C and CM groups: nuclei (blue, A1,A2), CK-18 (red, B1,B2), ACOT7 (green, C1,C2), merged with CK18 and ACOT7 (D1,D2) staining, respectively. (E) Positive IF signals of ACOT7 protein quantified using ImageJ software. Con/C, control group. CM, clinical mastitis group. MA, mammary alveoli; MECs, mammary epithelial cells; Scale bars, 50 µm (200× magnification). ** represents p < 0.01.

Article Snippet: The slices were incubated with a rabbit anti-ACOT7 primary antibody at a 1:200 dilution (Proteintech, Wuhan, China).

Techniques: Staining, Software, Control

Journal: International journal of molecular sciences

Article Title: Role of Acyl-CoA Thioesterase 7 in Regulating Fatty Acid Metabolism and Its Contribution to the Onset and Progression of Bovine Clinical Mastitis.

doi: 10.3390/ijms252313046

Figure Lengend Snippet: Figure 6. Expression patterns of ACOT7 mRNA and protein in mammary glands of the two groups. (A) Relative expression level of ACOT7 mRNA, monitored via qRT-PCR assays. (B) Protein bands and relative expression level of ACOT7, monitored via Western blot assays and using ImageJ software, respectively. Con/C, control group. CM, clinical mastitis group. Data are presented as means ± SEM. ** represents p < 0.01.

Article Snippet: The slices were incubated with a rabbit anti-ACOT7 primary antibody at a 1:200 dilution (Proteintech, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: International journal of molecular sciences

Article Title: Role of Acyl-CoA Thioesterase 7 in Regulating Fatty Acid Metabolism and Its Contribution to the Onset and Progression of Bovine Clinical Mastitis.

doi: 10.3390/ijms252313046

Figure Lengend Snippet: Figure 7. Proposed molecular mechanism underlying the effects of ACOT7 in the mammary glands of dairy cows with clinical mastitis (CM). MECs, mammary epithelial cells; FABP, fatty-acid-binding protein; FATP, fatty acid transport protein; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; LPS, lipopolysaccharide. Scale bar, 50 µm (200× magnification).

Article Snippet: The slices were incubated with a rabbit anti-ACOT7 primary antibody at a 1:200 dilution (Proteintech, Wuhan, China).

Techniques: Binding Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Sorting of miR-200c from cells to exosomes via Rab1A in breast cancer. ( A ) Dig-conjugated miR-200c and let-7a complex pulled down from whole cell extracts of 4T1 lung metastasis using anti-digoxin beads and analyzed by electrophoresis followed by Coomassie blue staining. ( B ) MALDI-TOF-MS analysis of tryptic peptides from the band indicated by red arrows in ( A ). ( C ) Western blot analysis of Rab1A proteins from before (top panel) and after anti-Dig Alpha Donor beads pull-down (bottom panel) in 4T1 lung metastasis lysate. ( D ) ChIP analysis of the interaction between Rab1A and miR-200c using anti-Rab1A antibody pull-down exosomal RNAs and access the miR-200c with miRNA qPCR. ( E ) SPR analysis of the interaction between Rab1A recombinant protein and biotinylated (Bio-) miR-200c or miRNA scramble control covalently immobilized onto the streptavidin-coated sensor chip. ( F ) Rab1A knockout (KO) by transfection of Rab1A CRISPR/Cas9 plasmid with and without miR-200c inhibitor in 4T1 cells. qPCR analysis of miR-200c in exosome and donor cell 3 days after transfection. ( G ) Representative Western blot analysis. ( H ) Quantification of cell number after 48 h culture in 24-well plate. **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Electrophoresis, Staining, Western Blot, Recombinant, Control, Knock-Out, Transfection, CRISPR, Plasmid Preparation

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Increased miR-200c trafficking from cell to exosomes promotes breast cancer lung metastasis. ( A ) Representative IHC analysis of Rab1A level in Tri + (n=28) or Tri − (n=25) breast cancer tumor (T) specimens and adjacent normal tissues ( A ) (n=20). Scale bars, 50 μm. ( B ) Quantification of PD-L1 + cell ratio in the specimens. ( C ) Representative immunoblot analysis of Rab1A in the tumor tissues. ( D ) Analysis of miR-200c in lung metastasis using qPCR. ( E ) Representative 4T1 primary breast tumor (top left) and lung (bottom left) with metastatic nodules from tumor-bearing mice (n=5) at 28 days injected with 4T1 cells with Rab1A activation particles via intravenous injection (50 mg/kg, body weight, every other day). ( F ) Quantification of primary tumor volumes (left panel) and metastasis nodule number (>1 μm) (right panel). The tumor size was assessed by the following formula: volume = (width) 2 × length/2. ( G ) Representative immunoblots of Rab1A in tumors (left panel) and qPCR analysis of miR-200c in tumor exosomes (right panel). ( H ) Survival rate of mice in ( E ). *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Western Blot, Injection, Activation Assay

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: Inhibition of miR-200c sorting protein interferes with breast cancer lung metastasis. ( A ) Survival rate of 4T1 tumor-bearing mice (n=8) treated with different doses of anti-Rab1A antibody or normal lgG control via intravenous injection. ( B ) Representative 4T1 breast primary tumor (top panel) and lung (middle panel) with metastatic nodules from tumor-bearing mice at 28 days after intravenous injection of 4T1 cells with anti-Rab1A antibody (10 mg/kg, body weight, every other day). Quantification of primary tumor volume (bottom left) and metastasis nodule number (>1 μm) (bottom right). ( C ) qPCR analysis of miR-200c in tumor tissues and exosomes. ( D ) Survival rate of mice. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Inhibition, Control, Injection

Journal: Breast Cancer : Targets and Therapy

Article Title: Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis

doi: 10.2147/BCTT.S400974

Figure Lengend Snippet: 4T1 exosomes inhibit the immune response of immune cells in the lung. ( A ) 4T1-derived exosomes labeled with fluorescence dye PKH16 (Exo/PKH26) and administered to mice by intravenous injection. Immunofluorescence analysis of Exo/PKH26 and F4/80. Cell nuclei stained with DAPI. Scale bars, 50 μm. ( B ) 4T1 tumor-bearing mice (n=8) treated with 4T1-derived exosomes at 50 mg/kg for 3 days via intravenous injection. Concentration of IFNγ and IL-1β in monocytes of breast cancer lung metastasis assessed by flow cytometry. Numbers above the box indicate percent cells. ( C ) 4T1 tumor-bearing mice (n=8) treated with different doses of anti-Rab1A antibody via intravenous injection. Flow cytometry analysis of exosome uptake efficacy by monocytes from lungs. ( D ) Analysis of IFNγ in the lung by ELISA. ( E ) Analysis of IL-1β in the lung by ELISA. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments (error bars, SD).

Article Snippet: The role of Rab1A on the miR-200c distribution in breast cancer cells was further analyzed by knocking out (KO) the Rab1A gene by transfection of Rab1A CRISPR/Cas9 KO plasmid (Santa Cruz, sc-422550) followed by treatment with or without miR-200c inhibitor in 4T1 cells.

Techniques: Derivative Assay, Labeling, Fluorescence, Injection, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of scutellarein on the combination of CDC4 and RAGE proteins. (A) Western blot analysis was performed to investigate the effects of scutellarein (40 µ M) on the expression of ubiquitin-associated proteins, including CDC4, RCHY1, MDM2, CBL and Smurf1 in SW480 cells. (B) An IP assay was performed to detect the effects of scutellarein on the combination of CDC4 and RAGE in SW480 cells. (C) Representative data presenting the levels of ubiquitin-associated proteins (CDC4, RCHY1, MDM2, CBL and Smurf1) in T84 cells treated with scutellarein. (D) An IP assay was used to assess the effects of scutellarein on the combination of CDC4 and RAGE proteins in T84 cells. n=3. * P<0.05 vs. the control group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; IP, immunoprecipitation; RCHY1, ring finger and CHY zinc finger domain containing 1; MDM2, MDM2 proto-oncogene; CBL, Cbl proto-oncogene; Smurf1, SMAD specific ubiquitin protein ligase 1.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Control, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of CDC4/RAGE on the proliferation and apoptosis of SW480 and T84 cells. Following 24 h of cell transfection with OE-NC or OE-CDC4, (A) SW480 and (B) T84 cells were treated with CHX (100 µ g/ml) for 1, 2, 4, 8 or 24 h, and then a western blot assay was performed to detect the protein levels of RAGE. (C) A western blot assay was performed to investigate the ubiquitination of RAGE subsequent to SW480 and T84 cells being transfected with OE-CDC4 or OE-NC. Cell proliferation of (D) SW480 and (E) T84 cells was detected using MTT analysis once the cells were transfected with OE-CDC4, OE-CDC4+OE-PAGE or their negative control vector. (F) Effects of CDC4 and RAGE overexpression on the apoptosis of SW480 and T84 cells were analyzed using flow cytometry. n=3. * P<0.05 vs. control group; # P<0.05 vs. the OE-CDC4 group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; OE, overexpression; NC, negative control; CHX, cycloheximide.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Negative Control, Plasmid Preparation, Over Expression, Flow Cytometry, Control

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Scutellarein treatment decreased RAGE expression and induced cell apoptosis via increasing CDC4 expression. (A) Reverse transcription-quantitative PCR and (B) western blot assays were used to determine the knockdown efficiency of sh-CDC4 in SW480 and T84 cells. * P<0.05 vs. the sh-NC group. Next, SW480 and T84 cells were divided into three groups (control, scutellarein and scutellarein+sh-CDC4), and then submitted to the following assays. (C) Expression of RAGE was detected using a western blot assay. Protein stability of RAGE was detected by using the CHX reagent together with a western blot assay in (D) SW480 and (E) T84 cells. Levels of cleaved caspase3/7 and total caspase3/7 were determined using a western blot assay in (F) SW480 and (G) T84 cells. * P<0.05 vs. the control group; # P<0.05 vs. the scutellarein group. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4; sh-, short hairpin RNA; NC, negative control; CHX, cycloheximide.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Control, shRNA, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Effects of scutellarein/CDC4 on the tumor growth in a mice xenograft model with a T84 cell injection. (A) Images of tumors removed from the mice treated with scutellarein and scutellarein+sh-CDC4. Bar graph represents the relative weight of the solid tumor types. (B) Immunohistochemical staining was used to detect the expression of Ki-67 in the tumor tissues derived from different groups. (C) Hematoxylin and eosin staining was used to assess the pathological alteration of the tumor tissues derived from different groups. Data are presented as the mean ± standard error of the mean. n=10. * P<0.05 vs. the control group; # P<0.05 vs. the scutellarein group. CDC4, cell division control protein 4; sh-, short hairpin RNA.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing, Derivative Assay, Control, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Scutellarein inhibits the development of colon cancer via CDC4-mediated RAGE ubiquitination

doi: 10.3892/ijmm.2020.4496

Figure Lengend Snippet: Schematic diagram of the anti-tumor function of scutellarein in colon cancer. Scutellarein treatment reduced the expression of RAGE via the CDC4-mediated ubiquitination degradation pathway, which then inhibited cell proliferation, tumorigenesis and induced cell apoptosis in colon cancer. RAGE, receptor for advanced glycation end products; CDC4, cell division control protein 4.

Article Snippet: In addition, the RAGE clustered regularly interspaced short palindromic repeats (CRISPR) Activation Plasmid (cat. no. sc-400284-ACT; Santa Cruz Biotechnology, Inc.), and CDC4 CRISPR Activation Plasmid (cat. no. sc-401257-ACT; Santa Cruz Biotechnology, Inc.) were used to upregulate the expression of RAGE (OE-RAGE) and CDC4 (OE-CDC4) in colon cancer, and were transfected into cells using Lipo3000 (cat. no. L3000008; Thermo Fisher Scientific, Inc.) at 37°C for 48 h, in addition to the Control CRISPR/dCas9 Activation Plasmid (OE-NC; cat. no. sc-437275; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Ubiquitin Proteomics, Control

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: A . Slides without primary antibody served as the negative control in GC tissues, B, E . Typical immunohistologic features with Rap1GAP expression in para-carcinoma and GC tissues, the Rap1GAP staining localized predominantly in the cytoplasm. C, F . Typical immunohistologic features with E-cadherin expression in para-carcinoma and GC tissues. D . Immunostaining for MMP2 was performed in GC; Magnifications, ×200.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Negative Control, Expressing, Staining, Immunostaining

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Differences in Rap1GAP, E-cadherin and MMP2 between the cancer tissues and para-carcinoma tissues

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Rap1GAP and E-cadherin protein in GC cells were notably lower expression compared to 293T cells by western blotting. Conversely, MMP2 was upregulated in GC cells (*P < 0.05 and **P < 0.01)

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Correlations between the clinicopathologic variables with Rap1GAP, E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Association between Rap1GAP with E-cadherin and MMP2

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The cumulative overall survival differences between the patients with high and low levels of protein expression. The P value was obtained using the log-rank test of the difference. A . Rap1GAP; B . E-cadherin; C . MMP2.

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Univariate Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Multivariate Cox Proportional Hazards Analysis for Overall Survival

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques:

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: The GC cells were grown and transfected with the negative control (NC) or Rap1GAP CRISPR Activation Plasmid. Overexpression Rap1GAP improved the expression of E-cadherin (A, B) and, inversely, suppressed the expression of MMP2 (A, B) compared to NC group in mRNA level and protein level. (*P < 0.05 and **P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Transfection, Negative Control, CRISPR, Activation Assay, Plasmid Preparation, Over Expression, Expressing

Journal: Oncotarget

Article Title: Low expression of Rap1GAP is associated with epithelial-mesenchymal transition (EMT) and poor prognosis in gastric cancer

doi: 10.18632/oncotarget.14074

Figure Lengend Snippet: Overexpression Rap1GAP repressed the migration A . and invasion B . capacity of GC cells C . The graph shows the quantified data of the assay, migration (left) and invasion (right) (**P < 0.01).

Article Snippet: The GC cells were grown and transfected with the NC or Rap1GAP CRISPR Activation Plasmid (Santa Cruz) using Lipofectamine 2000, according to the manufacturer's protocol.

Techniques: Over Expression, Migration

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Temporal expression of endogenous MC1R and Nurr1 in the ipsilateral brain hemisphere post-HI. (a) Representative Western blot bands of the time course. (b) Western blot data showed that the endogenous expression levels of MC1R significantly increased from 12 h reaching peak at 48 h post-HI. (c) Nurr1 expression levels significantly increased over time, reached highest at the 48 h post-HI. n = 6 per group. Data were represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus 6 h HI, and & p < 0.05 versus 48 h HI; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Expressing, Western Blot, Hi-C

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Immunofluorescence staining showed MC1R and Nurr1 colocalization with neurons in the ipsilateral brain hemisphere at 48 h post-HI. Immunofluorescence staining showed that MC1R (a) and Nurr1 (b) expressions on neurons were seen to be higher in the vehicle-treated pup rats compared to the sham group, and a higher expression of MC1R (a) and Nurr1 (b) on neurons after BMS-470359 treatment compared with vehicle. Merge showed the colocalization of MC1R and Nurr1 on neurons. n = 2 per group. Neurons were stained red. MC1R and Nurr1 were stained green. DAPI was stained blue. Scale bar = 100 μ m.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Immunofluorescence, Staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: Effects of BMS-470539 treatment on neuronal apoptosis at 48 h post-HI, and the knockout efficiency of MC1R or Nurr1 knockout CRISPR in naive and HI rats. (a, b) Representative microphotographs and quantitative analysis of C-Cas 3-positive neurons in the ipsilateral hemisphere at 48 h post-HI. The number of C-Cas 3-positive neurons significantly increased in the vehicle group compared with the sham group and BMS-470539 treatment group. n = 4 per group. C-Cas 3 was green. Neurons were red. Blue was for DAPI. Scale bar = 100 μ m. (c, d) Representative Western blot bands and quantitative analysis of MC1R and Nurr1 protein levels in the ipsilateral hemisphere at 48 h post-HI. The expression of MC1R or Nurr1 was significantly reduced by MC1R or Nurr1 knockout CRISPR in naive and HI rats. n = 4 per group (data were represented as mean ± SD; ∗ p < 0.05 versus sham; # p <0.05 versus HI+vehicle; @ p < 0.05 versus naive+control CRISPR, & p < 0.05 versus HI+control CRISPR; one-way ANOVA, Tukey's post hoc test).

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 treatment exerted its antioxidative stress and antiapoptosis effects via MC1R/cAMP/PKA/Nurr1 signaling pathway at 48 h post-HI. (a) Representative Western blot bands. (b–i) Quantification of MC1R, cAMP, p-PKA, Nurr1, 4-HNE, HO-1, Bax, and Bcl-2 expression levels in the ipsilateral hemisphere at 48 h post-HI. BMS-470539 treatment significantly increased the protein levels of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2 but significantly decreased the expression of 4-HNE and Bax compared to the HI+vehicle group. Knockout of MC1R or Nurr1 with CRISPR interventions significantly abolished such effects of BMS-470539. n = 6 per group. Data was represented as mean ± SD. ∗ p < 0.05 versus sham, # p < 0.05 versus HI+vehicle, @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Western Blot, Expressing, Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration reduced neuronal degeneration at 48 h post-HI, which was reversed by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs and quantitative analysis of FJC-positive neurons in the ipsilateral hemisphere at 48 h post-HI. n = 6 per group. FJC was green. Blue was for DAPI. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model

doi: 10.1155/2022/4054938

Figure Lengend Snippet: BMS-470539 administration suppressed oxidative stress at 48 h post-HI, which was abolished by MC1R or Nurr1 knockout CRISPR. (a, b) Representative microphotographs of MitoSox (red) and 8-OHdG (green) staining in the ipsilateral hemisphere at 48 h post-HI. Blue was for DAPI. (c, d) Quantitative analysis of MitoSox- and 8-OHdG-positive cells. n = 6 per group. Scale bar = 100 μ m. Data was represented as mean ± SD. ∗ p < 0.05 versus sham; # p < 0.05 versus HI+vehicle; @ p < 0.05 HI+BMS-470539 or HI+BMS-470539+control CRISPR; one-way ANOVA, Tukey's post hoc test.

Article Snippet: MC1R CRISPR KO plasmid (Santa Cruz Biotechnology, USA), Nurr1 CRISPR KO plasmid (Santa Cruz Biotechnology, USA), or control CRISPR plasmid (Santa Cruz Biotechnology, USA) was given via intracerebroventricular injection at 48 h before HI induction according to our previous study [ ].

Techniques: Knock-Out, CRISPR, Staining, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: Clinical and molecular features of patients with tumor molecular profiling on biopsies obtained upon resistance to ALK inhibitors in the MATCH-R study.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Biomarker Discovery, Sequencing, RNA Sequencing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Diverse resistance mechanisms to the third-generation ALK inhibitor lorlatinib in ALK-rearranged lung cancer

doi: 10.1158/1078-0432.CCR-19-1104

Figure Lengend Snippet: NF2 loss of function mediates resistance to lorlatinib. A, Clinical course of patient MR135 and mutational profile of samples obtained on lorlatinib progression (PD, progressive Disease). B, Cell survival assay assessed with Cell Titer Glo of MR135 lorlatinib resistant cells from biopsy 1 (MR135-R1) treated for 7 days with the indicated concentrations of lorlatinib and vistusertib (AZD2014) alone or in combination. C, Immunoblot analysis from cell lysates of MR135-R1 treated for 24hs with the specified doses of lorlatinib, vistusertib (AZD2014) and ponatinib alone or in combination using indicated antibodies. D, Athymic nude mice bearing MR135-R2 PDX were administered lorlatinib or vistusertib 20 mg/kg orally. Tumor volumes, mean ±SD (n =8); (*** p < 0.001). E, Cell lysates from H3122 parental and H3122 cells with NF2 heterozygous deletions or homozygous deletions, generated by CRISPR-CAS9 gene editing, were immunoblotted to detect merlin expression. H3122 cells with bi-allelic NF2 knock-out lacked merlin expression. F, Cell survival assay of H3122 parental and H3122 NF2 knock-out (NF2 KO) cells treated with lorlatinib for 7 days. Cell survival was assessed by Cell Titer Glo. G, Cell proliferation assay of H3122 parental and H3122 NF2 KO cells untreated and treated with lorlatinib measured at baseline, day 2, day 5 and day 7. Cell viability was assessed with Cell Titer Glo. H, Caspase 3/7 activation (Caspase 3/7-Glo assay) relative to the number of live cells simultaneously assessed in the cell proliferation assay previously described. I, H3122 parental and NF2 KO cells were treated with the indicated doses of lorlatinib for 24hs. Cell lysates were immunoblotted to detect the selected proteins.

Article Snippet: NF2 gene knock-out was performed with the CRISPR/Cas9 KO Plasmid (h) from Santa Cruz Biotechnology (sc-400504).

Techniques: Clonogenic Cell Survival Assay, Western Blot, Generated, CRISPR, Expressing, Knock-Out, Proliferation Assay, Activation Assay, Glo Assay