Journal: International journal of molecular sciences

Article Title: Cerebroprotective Effect of 17β-Estradiol Replacement Therapy in Ovariectomy-Induced Post-Menopausal Rats Subjected to Ischemic Stroke: Role of MAPK/ERK1/2 Pathway and PI3K-Independent Akt Activation.

doi: 10.3390/ijms241814303

Figure Lengend Snippet: Figure 6. Estradiol increases the CTMP protein expression. Carboxy-terminal modulator protein (CTMP) protein expression in brains from placebo- and E2-treated ovariectomized female rats sub- jected to tMCAO. (A) Representative Western blots and (B) relative abundance of CTMP in whole-cell lysates of non-ischemic (NI) and ischemic (I) hemispheres. Two-way ANOVA: E2 vs. placebo, p = 0.08, F1,24 = 3.1). Post hoc Sidak’s multiple comparisons test: significantly different from non-ischemic placebo group (* p < 0.05). Data are mean ± SEM of individual data points (normalized to β-actin). E2, 17β-estradiol. tMCAO, transient middle cerebral artery occlusion.

Article Snippet: Aliquots of protein (40 μg) were dissolved in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA) under reducing conditions, loaded on 4–12% Bis-Tris gels (Invitrogen), subjected to SDS-PAGE and electrotransferred to 0.2 μm nitrocellulose membranes for immunolabeling using the following primary antibodies: (1) anti-cleaved caspase-3 (Asp175), rabbit polyclonal antibody that detects endogenous levels of the large fragment (17/19 KDa) of activated caspase-3 (#9661;1:500; Cell Signaling Technology, Inc., Beverly, MA, USA); (2) anti-phospho MAPK (pERK1/2) rabbit monoclonal antibody, which recognizes ERK1 and ERK2 that are phosphorylated on both a Thr202 and a Tyr204 residue (D13.14.4E; #4370;1:2000; Cell Signaling Technology); (3) anti-MAPK1/2 (ERK1/2) rabbit polyclonal antibody (#06-182; 1:5000; Millipore, Temecula, CA, USA); (4) anti-phospho BAD (pBAD) rabbit monoclonal antibody, which detects endogenous levels of BAD only when phosphorylated at Ser136 (D25H8; #4366;1:500; Cell Signaling Technology); (5) anti-Bad rabbit polyclonal antibody (#9292; 1:500; Cell Signaling Technology); (6) anti-phospho Akt (pAkt) mouse monoclonal antibody, which recognizes Akt only when phosphorylated at Ser473 (193H12; #4058; 1:000; Cell Signaling Technology); (7) anti-Akt (Akt, pan) rabbit monoclonal antibody (C67E7; #4691; 1:1000, Cell Signaling Technology); (8) anti-CTMP, rabbit polyclonal antibody, which detects endogenous levels of total CTMP protein (#4612; 1:500; Cell Signaling Technology); and (9) anti-β-actin mouse monoclonal antibody (Clone AC-15, #A5441; 1:10000; Sigma, Saint Louis, MI, USA).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: NDRG4 promotes myogenesis via Akt/CREB activation

doi: 10.18632/oncotarget.21591

Figure Lengend Snippet: (A) Western blot results showing the changes in phosphatase and tensin homolog (PTEN) and CTMP protein expression levels after NDRG4 knockdown. (B) Relative protein expression levels of CTMP and PTEN represented by the ratio of detected protein to GAPDH protein expression level after NDRG4 knockdown at D0, D2, and D4. (C) Co-immunoprecipitation results of the interaction between NDRG4 and CTMP in vivo . C2C12 cells were differentiated for 3 days, harvested, and pulled-down by antibodies. Lane 1: The cell lysate (Input) was used as a positive control for Western blotting analysis. Lane 2: Immunoprecipitation results with anti-IgG antibody as a negative control. Lane 3: Immunoprecipitation results with anti-NDRG4 antibody. (D) The subcellular localization of NDRG4 and CTMP demonstrated by immunofluorescence in C2C12 myotubes. C2C12 myotubes were stained with antibodies against NDRG4 (red), CTMP (green), and DAPI (blue). (E) NDRG4-knockdown C2C12 cells and control cells were lysed and immunoprecipitated with either CTMP antibody or control IgG. Immuno-complexes and input cell lysates were analyzed by Western blotting with anti-pAkt, anti-NDRG4, and anti-CTMP antibodies.

Article Snippet: The membranes were blocked with 5% non-fat milk for 2 h and incubated overnight at 4°C with primary antibodies against NDRG4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-166,917, 1:200 dilution), MyoG (Santa Cruz Biotechnology; sc-12,732, 1:200 dilution), MyHC (Santa Cruz Biotechnology; sc-376,157, 1:3000 dilution), CREB (Cell Signaling Technology, Danvers, MA, USA; 48H2, 1:2000 dilution), GAPDH (Boster, China; BM0627, 1:1000 dilution), MyoD (Santa Cruz Biotechnology; sc-377,186, 1:200 dilution), Akt (Cell Signaling Technology, Danvers, MA, USA; 40D4, 1:2000 dilution), pAkt (Cell Signaling Technology, Danvers, MA, USA; D9E, 1:1000 dilution), carboxyl-terminal modulator protein (CTMP) (Santa Cruz Biotechnology, CA, sc-390,353 1:200 dilution), PTEN (Cell Signaling Technology, Danvers, MA, USA; Y184, 1:1000 dilution), and phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA; 87G3), followed by incubation with goat anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology; sc-2005, 1:3000 dilution) and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology; sc-2004, 1:3000 dilution) secondary antibodies for 1 h at room temperature.

Techniques: Western Blot, Expressing, Knockdown, Immunoprecipitation, In Vivo, Positive Control, Negative Control, Immunofluorescence, Staining, Control