Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 4. Immunolocalization of (A-C) platelet-derived growth factor-B (PDGF-B) and (D-F) PDGF receptor-β (PDGFR-β) in brain tissue of (A,D) controls, (B,E) lipopolysaccharide (LPS) group at 72 h, and the (C,F) glutamine (Gln) treatment group at 72 h. PDGF-B immunoreactivity was observed in the cyto plasm, PDGFR-β immunoreactivity was observed in the membrane of cortical neurons.

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Derivative Assay, Membrane

Journal: Molecular medicine reports

Article Title: Protective effects of glutamine in a rat model of endotoxemia.

doi: 10.3892/mmr.2012.1007

Figure Lengend Snippet: Figure 6. Western blot analysis of PDGF receptor-β (PDGFR-β) protein at various time points following injection of lipopolysaccharide (LPS). Upper row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the treatment group as 6-10 (2, 6, 12, 24 and 72 h). Lower row depicts the control group from left to right as 1-5 (2, 6, 12, 24 and 72 h) and the LPS group as 6-10 (2, 6, 12, 24 and 72 h).

Article Snippet: Rabbit anti-rat NF-κB, PDGF-B, and PDGFR-β antibodies and ABC kits were purchased from Boster (Wuhan, China).

Techniques: Western Blot, Injection, Control

Journal: Alzheimer's Research & Therapy

Article Title: ApoA-I deficiency increases cortical amyloid deposition, cerebral amyloid angiopathy, cortical and hippocampal astrogliosis, and amyloid-associated astrocyte reactivity in APP/PS1 mice

doi: 10.1186/s13195-019-0497-9

Figure Lengend Snippet: Cortical levels of pro-inflammatory protein and mRNA markers were increased in the absence of apoA-I. a IL-1β, c PDGFRβ, and d VCAM-1 protein levels were measured by ELISA in soluble half-brain homogenates; values were normalized to total protein concentration in the homogenates. b Il1b mRNA expression was measured in the cortex by qRT-PCR and normalized to β-actin expression. Points represent individual mice, and bars represent mean values. Circles represent female mice, and squares represent male mice. Omnibus analyses of apoA-I and APP/PS1 genotype effects by two-way ANOVA are displayed as exact p values below graphs. Sidak’s multiple comparisons test results are displayed within graphs as * p < 0.05, *** p < 0.001, and **** p < 0.0001. For ELISA, N = 5–19 mice per genotype were used; for mRNA, N = 7–21 mice per genotype were used. apoA-I, apolipoprotein A-I; HEM, hemizygous apoA-I genotype; KO, knockout apoA-I genotype; WT, wildtype APP/PS1 genotype; APP/PS1, transgenic APP/PS1 genotype; IL-1β, interleukin 1 beta; VCAM-1, vascular cell adhesion molecule 1; PDGFRβ, platelet-derived growth factor receptor beta

Article Snippet: Murine GFAP (NS830, Millipore, 1:400), intercellular adhesion molecule 1 (ICAM-1) (ab100688, Abcam, 1:2), vascular cell adhesion molecule (VCAM-1) (ab100750, Abcam, 1:2000), platelet-derived growth factor receptor beta (PDGFRβ) (MBS919047, MyBioSource, 1:10), interleukin 1 beta (IL-1β) (K15245D, MesoScale Discovery, 1:2), and tau (K15121D, MesoScale Discovery, 1:50) levels were measured in carbonate soluble half-brain lysates using commercial sandwich or MesoScale Discovery ELISA kits according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing, Quantitative RT-PCR, Knock-Out, Transgenic Assay, Derivative Assay

Journal: Cancer Science

Article Title: Simvastatin functions as a heat shock protein 90 inhibitor against triple‐negative breast cancer

doi: 10.1111/cas.13748

Figure Lengend Snippet: K292‐acetylated heat shock protein 90α (Hsp90α) is a direct target of simvastatin (Sim). Expression of Hsp90 clients was time‐dependently decreased in MDA ‐ MB ‐231 cells treated with 2 μmol/L Sim (A) or MDA ‐ MB ‐468 cells treated with 8 μmol/L Sim (B) for indicated times. Expression of Hsp90 clients in MDA ‐ MB ‐231 (C) or MDA ‐ MB ‐468 (D) cells were dose‐dependently decreased by treatment at indicated concentrations of Sim for 24 hours. E, LBH 589 (LBH) enhanced the complex formation of K292‐acetylated Hsp90α with its cochaperones in a dose‐dependent manner. Cell lysates from MDA ‐ MB ‐231 cells treated as indicated for 24 h were immunoprecipitated (IP) using acK292 antibody followed by immunoblotting (IB) with indicated antibodies. F, Acetylation status at K292 is the determinant for the complex formation of acetylated Hsp90 with its cochaperones and kinase clients. MDA ‐ MB ‐231 cells were transfected with Wt or K292R mutant of Hsp90 followed by the treatment as indicated for 24 h. Cell lysates were immunoprecipitated with M2 beads, and the coprecipitated proteins were detected by western blotting. G, Sim interfered with the colocalization of Hsp90 and Cdc37 in MDA ‐ MB ‐468 cells. Cells were incubated with monoclonal anti‐Hsp90 antibody and polyclonal andti‐Cdc37 antibody, and then with fluorescent antibodies. H, Sim binds preferentially with K292‐acetylated Hsp90α. Cell lysates from MDA ‐ MB ‐231 cells stably expressing Flag‐tagged Hsp90α treated with or without 25 nmol/L LBH 589 for 24 h were immunoprecipitated with acK292 antibody or M2 beads. Beads were washed, and incubated with 8 μmol/L Sim for 2 hours followed by digestion with indicated concentrations of trypsin for 5 min, and Hsp90α was detected by western blotting (WB). CDK4, cyclin‐dependent kinase 4; Ctl, control; eEF2K, Eukaryotic elongation factor 2 kinase; EGFR, epidermal growth factor receptor; HOP, Hsp70‐Hsp90 organizing protein 1; PDGFRβ, Platelet‐derived growth factor receptor β; p‐HSF1,S326 phosphorylated Heat shock factor 1; TGFβR2, TGF‐β receptor type 2

Article Snippet: The following reagents and antibodies were used: Cdc37, cyclin‐dependent kinase 4 (CDK4), Cyclin D1, Hsp27, p‐(S326)‐Heat shock factor 1, and Serine/threonine‐protein kinase 33 (Abcam, Cambridge, MA, USA); p21 Cip1 (BD Biosciences, Sparks, MD, USA); AKT, p‐AKT, caspase‐3, cleaved caspase‐3, caspase‐8, active caspase‐8, caspase‐9, cleaved caspase‐9, p‐Cdc37, CDK2, CDK6, Cyclin D1, Eukaryotic elongation factor 2 kinase, p‐eEF2K (S366), Hsp70‐Hsp90 organizing protein 1, HSF1, MEK1/2, p‐MEK1/2, Erk1/2, p‐Erk1/2, and poly(ADP‐ribose) polymerase (PARP)1 (Cell Signaling Technology, Danvers, MA, USA); Hsp40/Hdj1, Hsp70, Hsp90α, and p23 (Enzo Life Sciences, New York, NY, USA); Platelet‐derived growth factor receptor β (Merck Millipore, Temecula, CA, USA); Raf‐1 (C20) and Activator of Hsp90 ATPase protein 1 (Santa Cruz Biotechnology, Dallas, TX, USA); LBH589, Sim, mevastatin (Mev), pravastatin, anti‐Myc beads (Selleck Chemicals, Houston, TX, USA); mevalonate, anti‐Flag, β‐actin, anti‐M2, and anti‐HA beads (Sigma‐Aldrich, St. Louis, MO, USA); and Transforming growth factor‐β receptor II (Thermo Fisher Scientific, Grand Island, NY, USA).

Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Incubation, Stable Transfection, Derivative Assay

Journal: Cancer Science

Article Title: Simvastatin functions as a heat shock protein 90 inhibitor against triple‐negative breast cancer

doi: 10.1111/cas.13748

Figure Lengend Snippet: LBH 589 (LBH) enhances the inhibitory effects of simvastatin (Sim) on heat shock protein 90 (Hsp90) chaperone activity. Cell proliferation were assessed for triple negative breast cancer cells treated with Sim (A) or Sim plus 25 nmol/L LBH (B) for 72 hours using CCK ‐8 reagents. MDA ‐ MB ‐231 cells were treated as indicated for 24 hours followed by western blotting for Hsp90 client proteins associated with cell cycle (C) or protein kinase clients (E), and similarly, Hsp90 client proteins associated with cell cycle (D) or protein kinase clients (F) in MDA ‐ MB ‐468 cells. CDK, cyclin‐dependent kinase; PDGFRβ, Platelet‐derived growth factor receptor β; p‐HSF1, Heat shock factor protein 1; TGFβR2,TGF‐β receptor type 2

Article Snippet: The following reagents and antibodies were used: Cdc37, cyclin‐dependent kinase 4 (CDK4), Cyclin D1, Hsp27, p‐(S326)‐Heat shock factor 1, and Serine/threonine‐protein kinase 33 (Abcam, Cambridge, MA, USA); p21 Cip1 (BD Biosciences, Sparks, MD, USA); AKT, p‐AKT, caspase‐3, cleaved caspase‐3, caspase‐8, active caspase‐8, caspase‐9, cleaved caspase‐9, p‐Cdc37, CDK2, CDK6, Cyclin D1, Eukaryotic elongation factor 2 kinase, p‐eEF2K (S366), Hsp70‐Hsp90 organizing protein 1, HSF1, MEK1/2, p‐MEK1/2, Erk1/2, p‐Erk1/2, and poly(ADP‐ribose) polymerase (PARP)1 (Cell Signaling Technology, Danvers, MA, USA); Hsp40/Hdj1, Hsp70, Hsp90α, and p23 (Enzo Life Sciences, New York, NY, USA); Platelet‐derived growth factor receptor β (Merck Millipore, Temecula, CA, USA); Raf‐1 (C20) and Activator of Hsp90 ATPase protein 1 (Santa Cruz Biotechnology, Dallas, TX, USA); LBH589, Sim, mevastatin (Mev), pravastatin, anti‐Myc beads (Selleck Chemicals, Houston, TX, USA); mevalonate, anti‐Flag, β‐actin, anti‐M2, and anti‐HA beads (Sigma‐Aldrich, St. Louis, MO, USA); and Transforming growth factor‐β receptor II (Thermo Fisher Scientific, Grand Island, NY, USA).

Techniques: Activity Assay, CCK-8 Assay, Western Blot, Derivative Assay