Journal: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 1. Inhibition of micro RNA miR-122-5p on lipopolysaccharide-induced myocardial injury. Wistar rats were intravenously injected with miR-122-5p antagomir, followed by lipopolysaccharide (LPS) stimulation (n = 6 rats per group). (a-b) After LPS treatment for 12 h, heart tissues were harvested for the relative expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) using real-time quantitative PCR (RT-qPCR) or western blot assay. (c) The ratio of heart weight/ body weight (HW/BW) was calculated. (d) Hematein and eosin (H&E) staining revealed the effect of miR-122-5p on LPS-induced histopathological changes in heart. (e) Levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were examined by enzyme-linked immunosorbent assay (ELISA). ***p < 0.001 versus control; ##p < 0.01, ###p < 0.001 versus LPS + NC antagomir.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Inhibition, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 3. In vitro analysis for beneficial role of inhibiting micro RNA miR-122-5p in lipopolysaccharide (LPS)-induced apoptosis. (a-b) Rat H9c2 cells were treated with LPS for 12 h or 24 h, and the expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were assessed by real-time quantitative PCR (RT-qPCR) or western blot analysis. (c-d) H9c2 cells were transfected with NC inhibitor or miR-122-5p inhibitor for 24 h, followed by LPS treatment for another 24 h under proper culture conditions. After that, miR-122-5p and GIT1 expression levels were measured. (e) The contents of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected by appropriate kits. (f) Flow cytometry showed the apoptosis of LPS-stimulated myocardial cells. (g) Western blot analysis illustrated the changes of caspase-3 expression. **p < 0.01, ***p < 0.001 versus control; ++p < 0.01, ++

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 5. Potential downstream target gene of micro RNA miR-122-5p. H9c2 cells were transfected with NC mimics, miR-122-5p mimics, NC inhibitor and miR-122-5p inhibitor for 48 h. (a-b) The expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were verified by real-time quantitative PCR (RT-qPCR) assay. (c) The predicted binding sites of miR-122-5p in the 3-UTR of GIT1, and the sequence information of miR-122-5p and GIT1 (wild- or mutant- type) was displayed. (d) Luciferase assay verified the correlation between miR-122-5p and GIT1. aap < 0.01, aaap < 0.001 versus NC mimics; bbbp < 0.001 versus NC inhibitor; ddp < 0.01 versus NC mimics + GIT1 3UTR (WT).

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 6. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency attenuates the effects of micro RNA miR-122-5p loss on myocardial injury. (a) H9c2 cells were transfected with GIT1 siRNA to downregulate GIT1 expression. (b) The cells were transfected with GIT1 siRNA and/or miR-122-5p inhibitor, and then induced by lipopolysaccharide (LPS). GIT1 expression at mRNA and protein levels was then measured using real-time quantitative PCR (RT-qPCR) or western blot. (c) Apoptosis of myocardial cells was analyzed by flow cytometry. (d) Reactive oxygen species (ROS) production was examined using flow cytometry. (e-g) The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and tumor necrosis factor alpha (TNF-α) were assessed by the enzyme-linked immunosorbent assay (ELISA) kits. XXXp < 0.001 versus NC siRNA; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 7. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency inhibits nuclear factor erythroid 2-related factor 2 (Nrf-2) activation. (a) Real-time quantitative PCR (RT-qPCR) assay was used to measure the heme oxygenase-1 (HO-1) and NAD(p)H: quinone oxidoreductase 1 (NQO-1) expression. (b) Nuclear Nrf-2 level was revealed using western blot analysis. ^^p < 0.01 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Neuronal polarization in the developing cerebral cortex

doi: 10.3389/fnins.2015.00116

Figure Lengend Snippet: Regulation of cortical neuron polarization. Axon formation in vivo is influenced by extracellular cues. Intracellular effectors regulating cytoskeletal dynamics and membrane vesicle transport function in the initiation, stabilization, and subsequent elongation of a single axon.

Article Snippet: Cytoskeletal changes dependent on protein phosphorylation are required for axon specification; thus, axon formation can be assessed using markers recognizing differential phosphorylation states of cytoskeletal proteins (Sternberger and Sternberger, ; Mandell and Banker, ).

Techniques: In Vivo, Membrane

Journal: Database: The Journal of Biological Databases and Curation

Article Title: Phosprof: pathway analysis database of drug response based on phosphorylation activity measurements

doi: 10.1093/database/baac072

Figure Lengend Snippet: Scheme of the data collection and analysis for Phosprof. (Left) A drug from the Selleck L2400 Library was added to the cell culture media 2 h prior to the harvest of the cell lysate, which was then applied to a protein array with ATP. The tyrosine residues of the proteins on the array are phosphorylated by the cell lysate and detected using 4G10 antibody. (Right) The resultant ‘phosphorylation profiles’ were then analyzed to identify the significant pathways. The collected data can be browsed using various pathway analysis tools.

Article Snippet: Cell lysates including 100 μg of total protein were applied to an array with additional ATP at 30°C for 3 h. After the termination of the kinase reaction, the array was washed with Tris Buffered Saline with 0.05% Tween20 (TBST) and stained with the 4G10 phosphorylated protein-specific antibody (Merck, #05-1050) and a secondary fluorescein-conjugated antibody (Thermo, #A21235) to detect the phosphorylated tyrosine residues.

Techniques: Cell Culture, Protein Array

Journal: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: TRPC3 expression and SOCE in melanoma. a Representative images of immunohistochemical staining of HE, MART1, and TRPC3 in a melanoma primary tissue microarray (stage II) (original magnification, b ×200). The calibration bars represent 200 µm. b mRNA expression in various melanoma cell lines. SK-Mel-2 is a skin metastasis melanoma cell line with NRAS mutation, SK-Mel-24 is a lymph node metastasis cell line with BRAFV600E mutation, SK-Mel-187 is also a lymph node metastasis cell line, C8161 is a metastasis cell line with wild-type BRAF, and HEMA-LP is a skin melanocyte cell line. TRPC3 mRNA is widely expressed in human melanoma cell lines, independently of BRAF mutation. c Immunoprecipitation for TRPC3 and STIM1 was performed. d Cytosolic Ca2+ level in C8161 cells is shown as mean ± SD (n = 8–9). SOCE was examined in the presence or absence of DMSO (vehicle control 1 µM) or Pyr3 (1 µM) in C8161. Pyr3 inhibited SOCE in C8161 cells. Pyr3 was added after the addition of Ca2+. The Ca2+ signal was immediately decreased, indicating that Pyr3 inhibits SOCE in melanoma cells. Data in each panel are averages of eight or nine cells

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Mutagenesis, Immunoprecipitation, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: Pyr3 inhibits phosphorylation of STAT5 and Akt. a Representative images of Akt phosphorylation are shown. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of Akt was inhibited by TRPC3 inhibitor, Pyr3 (n = 4, **p < 0.01, ns not significant). b Protein phosphorylation microarray analysis in the presence of Pyr3. C8161 cells were incubated with DMSO (vehicle control) or Pyr3 (10 μM) for 15 min. The Y-axis shows the signal ratio of phosphorylated to non-phosphorylated protein in the presence of Pyr3 as a percentage of that of the DMSO control. c Representative images of STAT5 phosphorylation. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of STAT5 was inhibited by Pyr3 (n = 4, *p < 0.05, ns not significant)

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Phospho-proteomics, Western Blot, Microarray, Incubation, Control

Journal: Stem Cell Research & Therapy

Article Title: Effects of mesenchymal stem cells from different sources on the biological functions of multiple myeloma cells

doi: 10.1186/s13287-025-04222-8

Figure Lengend Snippet: Identification of SMSCs. ( A ) Schema of experiment design: MSCs induced senescence and identification. ( B ) SA-β-Gal staining of senescent and non-senescent MSCs. ( C ) Flow cytometry of senescent MSCs and non-senescent MSCs. ( D ) Levels of p21 and p53 protein in senescent MSCs and non-senescent MSCs. * P < 0.05, ** P < 0.01 comparison with the control group. Full-length gels/blots are shown in Figure in the Supplementary material

Article Snippet: 5% skim milk was used to block the membranes at room temperature for 1 h. The membranes were then incubated gently overnight at 4 °C with the following primary antibodies: anti-phosphorylated protein 53 (P53), antiglyceral- dehyde-3-phosphate dehydrogenase (GAPDH), anti-cyclin dependent kinase inhibitor 1 A (p21), anti-nuclear factor kappa B (NF-κB), antiphosphati dylinositol-4,5-bispho- sphate 3-kinase (PI3K), anti-phosphorylated (P)PI3K, anti-protein kinase B (AKT), antiPAKT (1:1,000; Beyotime), anti-cyclin dependent kinase 6 (CDK6), antiCyclinE1, anti-SRY-box transcription factor 2 (SOX2), anti-NANOG, antioctamer-bind -ing protein 4 (OCT4) (1:1,000; ProteinTech, Rosemont, IL, USA), anti-Vascular endothelial growth factor A (VEGFA), anti-interleukin-6 (IL6), anti-E-Cadherin and anti-N-Cadherin (1:1,000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Flow Cytometry, Comparison, Control

Journal: Stem Cell Research & Therapy

Article Title: Effects of mesenchymal stem cells from different sources on the biological functions of multiple myeloma cells

doi: 10.1186/s13287-025-04222-8

Figure Lengend Snippet: Effect of MSCs supernatants on the MM cells cycle and selected protein levels. ( A ) Schema of experiment design: The effects of BM-MSCs and UC-WJ MSCs supernatants on the cell cycle of RPMI8226 and U266 cells. ( B ) Levels of CDK6, P53 and CyclinE1 in MM cells after treatment with concentrated supernatants of MSCs. ( C ) Flowchart of the process of detecting intracellular cycle changes after the incorporation of different MSCs concentrates into MM cells.* P < 0.05, ** P < 0.01, *** P < 0.005 comparison with the control group. Full-length gels/blots are shown in Figure S2 in the Supplementary material

Article Snippet: 5% skim milk was used to block the membranes at room temperature for 1 h. The membranes were then incubated gently overnight at 4 °C with the following primary antibodies: anti-phosphorylated protein 53 (P53), antiglyceral- dehyde-3-phosphate dehydrogenase (GAPDH), anti-cyclin dependent kinase inhibitor 1 A (p21), anti-nuclear factor kappa B (NF-κB), antiphosphati dylinositol-4,5-bispho- sphate 3-kinase (PI3K), anti-phosphorylated (P)PI3K, anti-protein kinase B (AKT), antiPAKT (1:1,000; Beyotime), anti-cyclin dependent kinase 6 (CDK6), antiCyclinE1, anti-SRY-box transcription factor 2 (SOX2), anti-NANOG, antioctamer-bind -ing protein 4 (OCT4) (1:1,000; ProteinTech, Rosemont, IL, USA), anti-Vascular endothelial growth factor A (VEGFA), anti-interleukin-6 (IL6), anti-E-Cadherin and anti-N-Cadherin (1:1,000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Comparison, Control

Journal: Oncotarget

Article Title: Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

doi: 10.18632/oncotarget.26907

Figure Lengend Snippet: ( A ) Shown are sample two receptor-based three-point pharmacophore models generated on the RACK1A phosphorylation site with exclusion spheres colored pink, geometric and distance constraints (flexible) shown as lines and filled white circles as centers. HP-hydrophobic; D-donor; A-acceptor. ( B ) Ligand-based pharmacophore model generated on SD-29 with pharmacophore constraints acceptor, donor, hydrophobic ring, and hydrophilic sites represented filled circles. Structures of compounds SD-29-12 and SD-29-14 are shown.

Article Snippet: Twenty five microgram of proteins were loaded on the BioRad’s 4-12% precast Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane and then blocked with 5% Bovine Serum Albumin (BSA) for one hour, washed and incubated with the an antibody (1:100 dilution) to detect phosphorylated Y248 residue of RACK1A protein which was raised using the epitope: FSPNR{pTYR}WLCAATEH (Genscript, Piscataway, NJ, USA).

Techniques: Generated, Phospho-proteomics

Journal: Oncotarget

Article Title: Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

doi: 10.18632/oncotarget.26907

Figure Lengend Snippet: ( A ) Docked Model of RACK1A with SD-29 at the Y248 phosphorylation site. (left panel) Modeled structure of RACK1A with SD-29 (carbon in green) docked into it. The targeted binding pocket is highlighted in green. RACK1A is shown as ribbon model (white). (right panel) Detailed view of the SD-29 (carbon in green) interaction with RACK1A site pocket. The residues interacting with SD-29 are shown in a ball-and-stick model. Hydrogen bonds are shown as red broken lines. SD-29 binding site is surrounded by both hydrophobic (HP1) and hydrophilic residues (HP2). The structural model of ‘SD-29’ with RACK1A showing hydrogen bonds with Ser244, Trp249 and hydrophobic interactions with Tyr248, Phe243, Pro204, Leu 263and Trp249 residues. ( B ) RACK1 functional inhibitor compounds inhibit stress hormone induced RACK1A Y248 phosphorylation. One-week old Arabidopsis seedlings were treated with 10 μM of stress hormone Abscisic acid (ABA) in the presence/absence of the inhibitor compounds for 12 hours in a growth chamber (overnight) at 22 ° C. Lysates were probed with an antibody raised to detect phosphorylated Y248 residue of RACK1A protein in Arabidopsis . Lysates from a rack1a-1 knock-out mutant seedlings grown and treated similarly as the Wild Type seedlings were used as negative control. The compounds were dissolved in DMSO (D) and ABA was dissolved in methanol (M). The lower panel shows the same membrane stripped with stripping buffer and then probed with an Arabidopsis Actin antibody to show the loading control. ( C ) Salt stress-induced upregulation of RACK1 expression was inhibited by SD-29. The abundant leaf protein Rubisco large subunit (RbcL) was used as loading control for the blot. The 37kD RACK1 band was absent from the genetic knockout of RACK1 plants (double mutant- rack1ab lane). ( D ) Purified RACK1 protein on a SDS-PAGE gel. E. coli BL21(DE3) host strain was transformed with recombinant plasmid containing rice RACK1 (Chr05 Os05g47890) cDNA with a 3’ His tag. PMSF-induced bacterial lysate eluted from the glutathione-resin column was resolved by the SDS-PAGE electrophoresis for purity check. Lane M: Protein Marker; Lane S: Supernatant; Lane F: Flow through of supernatant; Lane W: Wash; and Lane E1~4: Elutions. ( E ) In the SPR assay, SD-29 (left panel) and SD-29-12 (right panel) bind directly to immobilized RACK1A on the surface of the chip via similar patterns, as evident in the sensogram. SD-29 (left panel) and SD-29-12 (right panel) were separately injected three times on the CM5 chip at 0, 1.56 μM, at 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM (top sensor) concentrations (left panel) and at 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM (top sensor) concentrations (right panel).

Article Snippet: Twenty five microgram of proteins were loaded on the BioRad’s 4-12% precast Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane and then blocked with 5% Bovine Serum Albumin (BSA) for one hour, washed and incubated with the an antibody (1:100 dilution) to detect phosphorylated Y248 residue of RACK1A protein which was raised using the epitope: FSPNR{pTYR}WLCAATEH (Genscript, Piscataway, NJ, USA).

Techniques: Phospho-proteomics, Binding Assay, Functional Assay, Residue, Knock-Out, Mutagenesis, Negative Control, Membrane, Stripping Membranes, Control, Expressing, Purification, SDS Page, Transformation Assay, Recombinant, Plasmid Preparation, Electrophoresis, Marker, SPR Assay, Injection