Journal: The Journal of Biological Chemistry

Article Title: The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER

doi: 10.1016/j.jbc.2022.102813

Figure Lengend Snippet: Phosphorylated serine 15 in the deduced 14-3-3 binding motif is required for potent binding to 14-3-3γ . A , the N-terminal region of TMCC3 highlighting the deduced 14-3-3 binding motifs. Serine residues being the possible phosphorylation sites in three deduced 14-3-3 binding motifs are indicated in red . Arginine, serine, and proline residues at −3, 0, and +2 positions, respectively, in the deduced 14-3-3 binding motif predicted with the highest score are underlined. B , serine 15 is required for potent binding to 14-3-3γ. Indicated combinations of HA-TMCC3-D1, HA-TMCC3-D1-S15A, HA-TMCC3-D1-S15/25/46A, and Flag-14-3-3γ were transfected into HEK293 cells, followed by immunoprecipitation with the anti-Flag mAb. The samples were immunoblotted with the anti-HA mAb and the anti-Flag pAb. The arrowheads indicate the bands of interest. The asterisk s indicate the nonspecific bands from the light chain of IgG. C , serine 15 is phosphorylated. HA-TMCC3-D1, HA-TMCC3-D1-S15A, and HA-TMCC3-D1-S15/25/46A were transfected into HEK293 cells, followed by immunoprecipitation with the anti-HA mAb. The samples were immunoblotted with the anti-phospho-14-3-3 binding motif pAb and the anti-HA pAb. The asterisk indicates the nonspecific bands from the light chain of IgG. TMCC3, transmembrane and coiled-coil domain family 3.

Article Snippet: The antibodies were purchased from the following commercial sources: mouse anti-HA mAb (BioLegend; Cat. No. 901514), rabbit anti-HA pAb (Sigma-Aldrich; Cat. No. H6908), rat anti-HA mAb (Roche; Cat. No. 11867423001), mouse anti-FLAG mAb (Sigma-Aldrich; Cat. No. F1804), rabbit anti-FLAG pAb (Sigma-Aldrich; Cat. No. F7425), rabbit anti-TMCC3 pAb (Sigma-Aldrich; Cat. No. HPA014272), rabbit anti-CLIMP-63 pAb (Bethyl Laboratories; Cat. No. A302–257A), mouse anti-α-tubulin mAb (Sigma-Aldrich; Cat. No. T6199), mouse anti-14-3-3 mAb (Santa Cruz Biotechnology; Cat. No. sc-1657), rabbit anti-phospho 14-3-3 binding motif pAb (Cell Signaling; Cat. No. 9601), mouse anti-PDI mAb (Abcam; Cat. No. ab2792), rat anti-GFP mAb (Nacalai; Cat. No. 04404–84), rabbit anti-GFP pAb (MBL; Cat. No. 598), and mouse anti-GAPDH mAb-HRP-DirecT (MBL; Cat. No. M171–7).

Techniques: Binding Assay, Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Caffeic Acid Derivatives Inhibit the Growth of Colon Cancer: Involvement of the PI3-K/Akt and AMPK Signaling Pathways

doi: 10.1371/journal.pone.0099631

Figure Lengend Snippet: (A) Nuclear proteins from tumor tissues were prepared for Western blotting analysis using monoclonal antibodies against p21 CIP1/WAF1 , cyclin D1, cyclin E, Cdk4 and c-myc as described under . The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The levels of detection represent the amount of these proteins in the nuclei of CRC cells in the experimental animals. The mean integrated densities of these proteins are adjusted with the control protein and shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk represent a statistically significant difference compared to the control group, P<0.05. (B) Cytoplasmic proteins from tumor tissues were prepared for Western blotting analysis using monoclonal antibodies against E-cadherin, N-cadherin, p-Akt, p-mTOR, p-ERK 1/2, p-AMPK, FASN and actin, as described under . The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The levels of detection represent the amount of these proteins in the cytoplasm of CRC cells in the experimental animals. The mean integrated densities of these proteins are adjusted with the control protein and shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk represent a statistically significant difference compared to the control group, P<0.05.

Article Snippet: The following monoclonal antibodies were purchased from Cell Signaling Technology, Inc.: Anti- N-cadherin (#4061), PTEN (#9559), anti-phosphorylation PDK1 (Ser241; #3061), total-PDK1(#3062), anti-phosphorylation Akt (S473; #4060), total-Akt (#9272), anti-phosphorylation GSK3α (S21; #9327), total- GSK3α (4337), anti-phosphorylation GSK3β (S9; #9323), total- GSK3β (#9315), anti-phosphorylation FOXO3 (T32; #9464), total- FOXO3 (#12829), total- TSC1 (#6935), total- TSC2 (#3990), total- LKB1 (#3047), total- 14-3-3 (#8312),anti-phosphorylation ERK 1/2 (T202/Y204; #9101), total-ERK 1/2 (#9102), anti-phosphorylation AMPKα (T172; #2535), total-AMPKα (#5832), anti-phosphorylation m-TOR (S2448; #5536), total-m-TOR (2983), anti-FASN(#3180), anti-NF-κB (p65) (#3033), anti-Cdk4(#2906), anti-p21 waf/cip1 (#2947), anti-cyclin E(#4132), anti-cyclin D1(#2978), anti-c-myc (#9402) and anti-Lamin A (#2032) (Danvers, MA).

Techniques: Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: The HDAC Inhibitor LBH589 Induces ERK-Dependent Prometaphase Arrest in Prostate Cancer via HDAC6 Inactivation and Down-Regulation

doi: 10.1371/journal.pone.0073401

Figure Lengend Snippet: ( A ) LBH589 induced Cdc25C-S216 dephosphorylation in a dose- and time-dependent manner. LNCaP was exposed to 75 nM LBH589 for various times or at indicated LBH589 concentrations for 24 h. Phosphorylation of Cdc25C-S216 was performed by immuno-blotting with Pi-Cdc25C-S216 antibody. ( B – C ) LBH589 treatment switched the PP1α interacting partner. The immuno-precipitations were conducted with anti-HDAC6 or anti-14-3-3ζ. The precipitated samples were analyzed by immuno-blotting using antibody against PP1α, 14-3-3ζ, Lys-Ac (Pan-acetylated lysine). IgG was a nonspecific pull-down control.

Article Snippet: The antibodies included HDAC1 (#2062), c-Raf (#9422), Akt (#9272), Pi-Akt (#4058), Pi-Cdc25C (Ser216) (#9528), Pi-c-Raf (S259) (# 9421), Pi-c-Raf (S338) (#9427), and Pi-Cdc2 (Tyr15) (#9111) purchased from Cell Signaling Technology (Beverly, MA, USA); HDAC6 (sc-28386), Pi-ERK(sc-7383), ERK (sc-94), 14-3-3ζ (sc-1019), and Cdc2 (sc-54) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Aurora A (#07-648), H3-Ac (#06-599), H4-Ac (#06-598), p21 (#05-345), and PP2A (#05-421) from Millipore (Lake Placid, NY, USA); GAPDH and actin (Sigma-Aldrich); Aurora B (#1788-1), Cdc25C (#1302-1), and PP1 (#1950-1) from Epitomics (Burlingame, CA); and HDAC3 (ab32369) from Abcam (Cambridge, UK).

Techniques: De-Phosphorylation Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Insulin Growth Factor 1 Protects Neural Stem Cells Against Apoptosis Induced by Hypoxia Through Akt/Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase (Akt/MAPK/ERK) Pathway in Hypoxia-Ishchemic Encephalopathy

doi: 10.12659/MSM.901055

Figure Lengend Snippet: Insulin growth factor 1 (IGF-1) activated the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. Neural stem cells (NSCs) were divided into four groups: the Control, Hypoxia, Hypoxia+IGF-1, and Hypoxia+IGF-1+PPP groups. ( A ) Protein expression levels of kinases were assayed by Western blot analysis. ( B ) The band intensity was assessed by Image Lab™ Software. The phosphorylation rate was expressed as the relative intensity of phosphorylated kinases/kinases, and the final results were normalized by GAPDH. Data presented are the mean ±SD of three independent experiments. * P <0.05; ** P <0.01. The significance marked at the top of the columns refers to comparison with the control group.

Article Snippet: After three rounds of rinsing with TBST, the membranes were incubated overnight at 4°C with primary antibodies against mammalian B cell lymphoma-2 (Bcl-2, 15071), Bcl-2-associated X protein (Bax, 14796), cytochrome c (11940), cleaved capase-3 (9661), pro capase-3 (9662), GAPDH (2118), phosphorylated mitogen-activated protein kinase (p-MAPK, 4370), MAPK (4695) (all from Cell Signaling Technology, Danvers, Massachusetts, USA), phosphorylated AKT (p-AKT, sc-101629), AKT (sc-8312), phosphorylated extracellular signal-regulated kinase (p-ERK, sc-16981-R), and ERK (sc-292838) (all from Santa Cruz Biotechnology, California, USA).

Techniques: Expressing, Western Blot, Software