Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes

doi: 10.1371/journal.pone.0111767

Figure Lengend Snippet: hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.

Article Snippet: 10 mg total protein was immunoaffinity-purified with anti-phospho-RXX(s/t) antibody for Akt substrates (CST 9614) or anti-phospho-(s)Q antibody for ATM/ATR substrates.

Techniques: Western Blot

Journal: International Journal of Biological Sciences

Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

doi:

Figure Lengend Snippet: Effect of IGF-1 on AKT/GSK-3β activity and cyclin D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).

Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

Techniques: Activity Assay, Expressing, Immunoprecipitation, Incubation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

doi:

Figure Lengend Snippet: Effect of Akt and GSK-3β inhibition on CREB phosphorylation and cyclin D1 and fibronectin expression. Mesangial cells were grown to 70% confluence and serum-starved overnight. Inhibitors for Akt/PKB (2 μM, SH-5) and GSK-β (100 nM, SB216763) were pre-treated for 30 min before adding IGF-1 (100 ng/ml) for 2 h. Cell extracts (30 μg protein) were subjected to Western blotting for (A) phosphorylated and total CREB and (B) for cyclin D1. Lane 1. Control; lane 2. IGF-1, 2h; lane 3. IGF-1, 2h+SH-5 (2 μM); and lane 4. IGF-1, 2h+SB216763 (100 nM). Representative blots from three different experiments are shown. (C). Effect of IGF-1 (100 ng/ml) or SB216763 (100 nM) on fibronectin expression upon 24 h exposure was determined by immunofluorescence microscopy. Nuclear staining was performed with Hoechst Dye 33342 and fibronectin with Alexis 488 (n=3).

Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining

Journal: International Journal of Biological Sciences

Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

doi:

Figure Lengend Snippet: IGF-1 activation of Akt substrate phosphorylation and interaction of phospho-GSK-3β with 14-3-3ζ. Mesangial cells were grown to 70-80% confluence and serum-starved overnight. IGF-1 (100 ng/ml) was added for 2 h and cells were harvested as described in Methods. Total extracts were subjected to SDS-PAGE and Western blotting with phospho-Akt motif antibodies (Figure A, left panel). 14-3-3ζ immunoprecipitates were probed with the phospho-Akt motif antibodies (Figure A, right panel). In addition, the 14-3-3 ζ immunoprecipitates were subjected to Western blotting for 14-3-3 ζ, GSK-3α/β, phosphorylated GSK-3β(S9P) and β-catenin (Figure B). The blots are representatives of three repeats.

Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

Techniques: Activation Assay, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks

doi: 10.3390/ijms22105245

Figure Lengend Snippet: Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C (PKC) activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and anti-β-catenin antibodies were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.

Article Snippet: Membranes were probed with the following antibodies (1:1000 dilution): phospho-PKC substrate motif ((R/K)XPSX(R/K)) MultiMabTM (#6967), MARCKS (#5607), Phospho-MARCKS (Ser167/170) (8722), p38 (#9212), c-Myc (#13987), myelin basic protein (#78896), β-catenin (#8480), phospho-ERK1/2 (#4370), ERK1/2 (#4695), phospho-AKT substrate (RXXS*/T*) (#9614S), PKCα (#9960), SQSTM1/p62 (#39749) were from Cell Signaling.

Techniques: Activation Assay, Western Blot, Cell Culture, Microscopy, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks

doi: 10.3390/ijms22105245

Figure Lengend Snippet: Signaling pathways and transcriptional factors modulated by PKC activation. ( A ) “Regulation of actin cytoskeleton” KEGG pathway is visualized. Red stars indicated differentially expressed proteins involved in the pathway and identified by MS/MS analysis, or Western blotting, after PMA treatment. ( B ) Expression profile of cofilin and phospho-cofilin was detected by Western blot at 0 h, 24 h, 48 h, 72 h and 96 h after treatment with PMA. Histogram represents the expression ratio of phospho-cofilin of the treated condition versus CTR sample. *** p -value < 0.001. ( C ) Phase-contrast images of MO3.13 cells cultured in a normal condition medium or treated with 100 nM PMA and 10 μM Y-27632, alone or in combination, for 96 h. Scale bar 200 μm. ( D ) Western blot analysis showing the activation level of PKC substrates in control and treated conditions after 1 h of stimulation. PMA at 100 nM and Y-27632 at 10 μM were used alone or in combination. The detection was performed through a phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody. The Ponceau S stained membrane is shown to verify the protein loading. ( E ) Western blot analysis showing the expression levels of cofilin and phospho-cofilin after treatment with PMA and Y-27632, used alone or in combination, for 96 h. Histogram represents the expression ratio of phospho-cofilin in the treated conditions versus the CTR sample. *** p -value < 0.001. ( F ) Hierarchical clustering based on Euclidean distance showing the separation between the CTR cells and those treated with PMA and Y-27632, alone and in combination. Profile plots of selected protein groups are shown, displaying distinct behavior concerning treatment.

Article Snippet: Membranes were probed with the following antibodies (1:1000 dilution): phospho-PKC substrate motif ((R/K)XPSX(R/K)) MultiMabTM (#6967), MARCKS (#5607), Phospho-MARCKS (Ser167/170) (8722), p38 (#9212), c-Myc (#13987), myelin basic protein (#78896), β-catenin (#8480), phospho-ERK1/2 (#4370), ERK1/2 (#4695), phospho-AKT substrate (RXXS*/T*) (#9614S), PKCα (#9960), SQSTM1/p62 (#39749) were from Cell Signaling.

Techniques: Activation Assay, Tandem Mass Spectroscopy, Western Blot, Expressing, Cell Culture, Staining