Journal: The Journal of Biological Chemistry

Article Title: Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

doi: 10.1074/jbc.M116.773739

Figure Lengend Snippet: Increased activity of SGK1 promotes pinocytosis in Akt3−/− macrophages. A, phosphorylation of Akt (Ser-473/Thr-308), total Akt, and Akt1/2 expression in WT and Akt3−/− MPMs treated with 50 ng/ml M-CSF. Ctl, control without M-CSF treatment. n = 3. B, uptake of Lucifer yellow dye by WT and Akt3−/− MPMs in the presence of 10 μm GSK3 inhibitor (SB), 20 nm mTOR inhibitor (RAPA), or 2 μm NFκB inhibitor (BAY). n = 8. C, phosphorylation and expression of NDRG1, an SGK1 specific substrate, in WT and Akt3−/− MPMs assessed by Western blotting. Bottom panel, expression of SGK1 in WT and Akt3−/− MPMs. n = 3. D, Lucifer yellow uptake by MPMs in the presence of SGK1 inhibitor (K-650394). n = 8. E, foam cell formation assay in WT and Akt3−/− MPMs cultured in the presence of 1 mg/ml LDL and in the presence or absence of 25 μg/ml SGK1 inhibitor (SGK1i) for 24 h (n = 8). F, effect of SGK1 inhibitor (GSK-650394, 25 μg/ml) on the uptake of Lucifer yellow by WT and Akt3−/− MPMs (n = 7). G, effect of Akt3 siRNA treatment on phosphorylation of NDRG1 and expression of NDRG1, Akt3, and Cdc42 in human MDMs assessed by Western blotting. GAPDH expression was used as a loading control (n = 6). H, uptake of Lucifer yellow dye by human MDM treated with control siRNA or Akt3 siRNA in the presence of 25 μg/ml SGK1i (n = 6). I, effect of 25 μg/ml SGKi on cholesterol accumulation in human MDM (n = 6). Human MDM were treated with Akt3 or control siRNA treatment and then cultured in the presence of 1 mg/ml LDL and 25 μg/ml SGK1i overnight, and cholesterol content was quantified. Data represent means ± S.E. *, p < 0.05. Data are representative of at least three independent experiments and are quantified from at least three independent experiments.

Article Snippet: Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) ( n = 3).

Techniques: Activity Assay, Expressing, Control, Western Blot, Tube Formation Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

doi: 10.1074/jbc.M116.773739

Figure Lengend Snippet: Akt3 inhibits macrophage pinocytosis via the SGK1/Cdc42 pathway. A, uptake of Lucifer yellow by WT and Akt3−/− MPMs in the presence or absence of 10 μm Cdc42 inhibitor (ML141). n = 8. B, uptake of Lucifer yellow dye by human MDMs treated with control siRNA or Akt3 siRNA in the presence of 10 μm ML141 (n = 6). C, cellular cholesterol levels of human MDMs treated with Akt3 siRNA or control siRNA in the presence of 1 mg/ml LDL and 10 μm ML141 (n = 6). D, Cdc42 activity in WT and Akt3−/− MPMs. Macrophages were treated with 25 μg/ml SGK1i overnight. Cells were then washed and lysed. Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) (n = 3). E, expression of Cdc42 in WT and Akt3−/− MPMs cultured in the presence or absence of 25 μg/ml SGK1i or 1 mg/ml LDL (n = 3). F, effect of suppression of Akt3 expression (siRNA treatment for 48 h, Akt3i) on Cdc42 expression in human MDM as assessed by Western blot analysis. GAPDH expression was used as a loading control (n = 6). G, expression of Rac1 in macrophages treated with 25 μg/ml SGK1 inhibitor and 0.8 mg/ml LDL (top panel). (n = 3). H, effect of SGK1i on RhoA expression in WT and Akt3−/− MPMs. Cells were incubated with or without 25 μg/ml SGK1i overnight. Cell protein samples were assessed by Western blot analysis. I, F-actin formation in WT and Akt3−/− MPMs exposed to 500 μ/ml LDL in the presence or absence (control) of SGK1 inhibitor. Center panels, F-actin formation (white arrows) at higher magnification. The actin cytoskeleton and the nuclei were visualized by staining with Alexa Fluor 488-phalloidin and DAPI, respectively. Scale bars = 25 μm, n = 8. Right panels, quantification of phalloidin fluorescence intensity. J, macrophages treated with 500 μg/ml LDL in the presence or absence of 25 μg/ml SGK inhibitor (GSK650394) for 4 h. Cells were stained with 0.6 μm TRITC-phalloidin. TRITC-phalloidin·F-actin complexes were extracted, and fluorescence was measured by SPECTRAmax GEMINI XS. Data represent means ± S.E. *, p < 0.05. Data are representative of at least three independent experiments.

Article Snippet: Cdc42 activity in cell lysates was measured using a RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton) ( n = 3).

Techniques: Control, Activity Assay, Activation Assay, Expressing, Cell Culture, Western Blot, Incubation, Staining, Fluorescence

Journal: The American journal of pathology

Article Title: Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

doi: 10.1016/j.ajpath.2013.08.026

Figure Lengend Snippet: Figure 2 PKCl/i deficiency influences activation of small GTPases. A: Protein lysates of deficient and control podocytes were used in activity assays for the small GTPases RhoA, Rac1, and Cdc42 and were analyzed using Western blot analysis. For loading control, the blotted membranes were incubated with GAPDH. B: Quantification of active GTPase versus total GTPase expression levels of RhoA, Rac1, and Cdc42. At least three different experiments of independent cell clones were used for each quantification. *P < 0.05, **P < 0.01.

Article Snippet: Activity Assays For activity measurements of the small GTPases, we used Rac1, Rho, and Cdc42 Activation Assay Biochem Kits (Cytoskeleton).

Techniques: Activation Assay, Control, Activity Assay, Western Blot, Incubation, Expressing, Clone Assay

Journal: The American journal of pathology

Article Title: Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

doi: 10.1016/j.ajpath.2013.08.026

Figure Lengend Snippet: Figure 7 Proposed model for influence of PKCl/i via Def-6 on the actin cytoskeleton. Deficiency of PKCl/i leads to increased activation of Def-6 and thereby to imbalance of the small GTPases, in particular Rac1. Active Rac1 influences several actin cytoskeletaledependent mechanisms.

Article Snippet: Activity Assays For activity measurements of the small GTPases, we used Rac1, Rho, and Cdc42 Activation Assay Biochem Kits (Cytoskeleton).

Techniques: Activation Assay

Journal: eLife

Article Title: Kinesin-2 autoinhibition requires elbow phosphorylation

doi: 10.7554/eLife.103648

Figure Lengend Snippet: Sequence alignment of OSM-3 and other members in the kinesin-2 family (KIF3A, KIF3B, KIF3C, and KIF17) from different model organisms as indicated. This figure shows the alignment results around the elbow region. Green arrowheads indicate the four phosphorylated residues. The position of amino acids of OSM-3 is labeled at the top of the figure. Sequences were aligned using CLUSTAL O (1.2.4) and presented by ESPript 3.0.

Article Snippet: Microtubule-stimulated ATPase activity assays were performed with a commercial kit (HTS Kinesin ATPase Endpoint Assay Biochem Kit, Cytoskeleton Inc) following the manufacturer’s instructions.

Techniques: Sequencing, Labeling

Journal: eLife

Article Title: Kinesin-2 autoinhibition requires elbow phosphorylation

doi: 10.7554/eLife.103648

Figure Lengend Snippet: ( A ) Microtubule-stimulated ATPase activity of wild-type (WT) OSM-3 and mutants. G444E, the hyperactive positive control; KHC, kinesin heavy chain. Average activity of KHC was set to 100% and others were normalized to KHC. ( B ) Summary of the single-molecular assay and the microtubule gliding assay. R.D., rarely detected. N.A., not available. Data are [mean ± SD (number of events)]. ( C ) Velocity distributions of microtubule gliding assays of the indicated OSM-3 constructs. n , total events measured. v , μm s –1 , average velocity with standard deviation. ( D ) Statistics of microtubule gliding velocities shown in ( C ). ( E ) Representative kymographs of the single-molecular movements of WT OSM-3 and mutants as indicated. Scale bars, vertical, 10 s; horizontal, 5 μm. ( F ) Velocity distributions of the single-molecular assays. n , total events measured. v , μm s –1 , average velocity with standard deviation. The distribution of G444E was fitted with a Gaussian distribution curve, while the distribution of phosphor-dead (PD) was fitted with a one-phase decay curve. ( G ) Run length distributions of the single-molecular assays. n , total events measured. l , average run length. The curves were fitted with the one-phase decay distribution. *p < 0.05, **p < 0.01, ****p < 0.0001, analyzed by one-way ANOVA, p values were adjusted by BH method.

Article Snippet: Microtubule-stimulated ATPase activity assays were performed with a commercial kit (HTS Kinesin ATPase Endpoint Assay Biochem Kit, Cytoskeleton Inc) following the manufacturer’s instructions.

Techniques: Activity Assay, Positive Control, Gliding Assay, Construct, Standard Deviation