Journal: BMC Cancer

Article Title: Protein kinase Cepsilon is important for migration of neuroblastoma cells

doi: 10.1186/1471-2407-8-365

Figure Lengend Snippet: Activation of PKC induces phosphorylation of MARCKS . (A) SK-N-BE(2)C cells were incubated in serum-free medium for 1 h followed by pre-treatment with 2 μM GF109203X, 2 μM Gö6976 or 200 nM LY333531 for 5 min and thereafter stimulation with 16 nM TPA. Cells were lysed and analysed with Western blot using antibodies detecting phosphorylated (p-MARCKS) and total MARCKS (tot-MARCKS). (B) Cells were transfected for three consecutive days with 50 nM of siRNA oligonucleotides against PKCα, PKCδ and PKCε or an equal amount of water. 18 h after the last transfection cells were incubated with serum-free medium and thereafter stimulated with 16 nM TPA for 1 h. Cells were lysed and analysed by Western blotting using antibodies against phosphorylated and total MARCKS.

Article Snippet: For detection, membranes were incubated with primary antibodies against phospho-MARCKS (1:500), phospho-Erk (1:500), Erk (1:500) (all Cell Signaling), MARCKS (1:1000) (Upstate), PKCα (1:3000), PKCβII (1:500), PKCδ (1:500) or PKCε (1:500) (all Santa Cruz Biotechnology) followed by incubation with a horseradish peroxidase-labelled secondary antibody (1:5000) (Amersham Biosciences).

Techniques: Activation Assay, Incubation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein

doi: 10.1371/journal.pone.0066512

Figure Lengend Snippet: (A and B) Adherent fibroblasts were stimulated with the indicated concentrations of PDGF-BB for the indicated times and Western Blot analysis for phosphorylated MARCKS (p-MARCKS), total MARCKS (MARCKS) and beta-actin was performed. (A) Representative dose-response (1 min stimulation) experiment. (B) Representative kinetics analysis (10 nM PDGF-BB) experiment. (C) Adherent fibroblasts were pretreated with 50 µM MANS, 50 µM RNS or PBS (VC for MANS or RNS peptides) for 30 minutes prior to stimulation with 10 nM PDGF-BB or VC (sterile water) for 1 minute. Western blot analysis was performed to determine the expression of phosphorylated MARCKS (p-MARCKS), total MARCKS (MARCKS), phosphorylated AKT1 (p-AKT), total AKT (AKT) and beta-actin. NT denotes no treatment and VC denotes vehicle control (sterile water); data is representative of three separate experiments.

Article Snippet: Phospho-MARCKS (Ser 152/156), phospho-AKT1 (Ser 473), total AKT1 and beta-actin antibodies were purchased from Cell Signaling Technology (Danvers MA) and total MARCKS (M-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Dysbindin Regulates the Transcriptional Level of Myristoylated Alanine-Rich Protein Kinase C Substrate via the Interaction with NF-YB in Mice Brain

doi: 10.1371/journal.pone.0008773

Figure Lengend Snippet: (A) SH-SY5Y cells were transfected with scrambled siRNA or siRNA for Dysbindin. Cell lysate of non-treated cells (Cont.), scrambled RNAi-transfected cells (Scr.) and RNAi for Dysbindin-transfected cells (siRNA) were subjected to Western blot with anti-MARCKS antibody. Columns and vertical bars denote the means ± SEM (triplicate independent experiments). Dysbindin knockdown cells exhibited significant reduction of MARCKS expression compared with control cells ( P <0.001, Student's t-test). (B) Hippocampus lysates were collected from wild-type mice or Dysbindin KO mice at P15, P20 and P45. The lysates were subjected to Western blot with anti-MARCKS antibody. Graphs and vertical bars denote the means ± SEM (triplicate independent experiments). At P45, Wild-type mice showed significant decreased MARCKS expression, while Dysbindin KO mice showed a maintained MARCKS expression. These data were confirmed by triplicate independent experiments (P<0.01, Student's t-test). ( C )Chromatin IP (ChIP) was performed using SH-SY5Y cells under the stimulation of retinoic acid. The promoter region of MARCKS was detected both in the IPs of anti-Dysbindin antibody (1 st panel) and those of anti-NF-YB antibody (2 nd panel), but not in the IPs of IgG (3 rd panel).

Article Snippet: Antibodies of anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag (Sigma-Aldrich, St Louis, MO), anti-V5 (Invitrogen), anti-β-actin (Chemicon International, Temecula, CA), anti-NF-YB (Santa Cruz Biotechnology), anti-MARCKS (Upstate), HRP-conjugated anti-mouse and Rabbit IgG (Cell Signaling Technology, Beverly, MA), and mouse normal IgG (Sigma-Aldrich) were purchased commercially.

Techniques: Transfection, Western Blot, Expressing, Chromatin Immunoprecipitation

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: PMA-induced ROS production is suppressed in monocytic MARCKS KO cells. ( A – D ) Monocytic THP-1-derived MARCKS WT and KO clones were differentiated with 100 nM calcitriol for 5 d. ROS production was induced by 100 nM PMA and assessed via luminol-amplified chemiluminescence in relative light units per second (RLU/s). ( A , B ) PMA-induced total ROS production is completely abolished in monocytic MARCKS KO cells. Kinetics ( A ) and the respective cumulated total ROS production (i.e., the area under the curve (AUC) within 60 min following PMA stimulation) ( B ) of total ROS production in MARCKS WT and KO clones (representative experiment, n = 3). ( C , D ) PMA-induced intracellular ROS production is strongly inhibited in MARCKS KO and partially reduced in MARCKS IM cells. Kinetics (( C ), representative experiment) and the respective cumulated intracellular ROS production within 60 min following PMA stimulation (( D ), mean ± SD, n = 3) in WT, IM, and KO clones. ( E ) Reconstitution of MARCKS in KO cells restores PMA-induced ROS production. MARCKS KO clones were differentiated with 100 nM calcitriol for 5 d. At day 3, cells were transfected with a MARCKS-expressing vector or the respective vector control (control cells: THP-1, transfection control: EGFP-encoding plasmid). Cumulated ROS production of EGFP-positive (i.e., transfected) cells within 60 min following PMA stimulation was detected by flow cytometry. The ROS production in control-transfected KO cells was set as 1 (mean ± SD, n = 3). The insets show the MARCKS levels in KO and THP-1 cells transfected with the control or the MARCKS expression vector. ( F ) Basal Akt levels are reduced and Akt re-phosphorylation is delayed in MARCKS KO cells. THP-1 WT and MARCKS KO cells were treated with 100 nM PMA. At the indicated time points, cells were harvested, and total cell extracts were prepared. Akt phosphorylation (at Thr308) and total Akt levels were determined via Western Blot (loading control: GAPDH; representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Derivative Assay, Clone Assay, Amplification, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Western Blot

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: ROS production is suppressed by PKCβ KO and PKC inhibitor Staurosporine, and MARCKS level and Ser167/170 phosphorylation are not affected in PKCβ KO cells. THP-1-derived PKCβ KO cells (generated using the CRISPR/Cas9 technique) and THP-1 WT cells were differentiated with 100 nM calcitriol for 5 d. Subsequently, ROS production was induced by PMA (100 nM) and determined (in RLU/s) via luminol-amplified chemiluminescence. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 60 min following stimulation ( B ) of total ROS generated by differentiated THP-1 WT and PKCβ KO cells (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 60 min following stimulation ( D ) of intracellular ROS generated by differentiated THP-1 and PKCβ KO cells (representative experiment, n = 3). ( E ) Primary human monocytes were incubated in medium with Staurosprine (Stauro; 80 nM) for 2.5 h. Subsequently, PMA-induced total ROS production was assessed and depicted as cumulated ROS production within 30 min following stimulation (mean ± SD; n = 2). ( F ) THP-1 WT and PKCβ KO cells were treated with 100 nM PMA. At the indicated time points, cells were harvested. Total cell extracts were prepared, and MARCKS ED phosphorylation (Ser167/170) and protein levels were determined (Western Blot; loading control: GAPDH; representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Derivative Assay, Generated, CRISPR, Amplification, Incubation, Western Blot

Journal: Antioxidants

Article Title: MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type

doi: 10.3390/antiox11081600

Figure Lengend Snippet: Increased monocytic ROS production following TNF long-term preincubation is dependent on MARCKS and PKCβ. ( A , B ) Kinetics ( A ) and the respective cumulated production (i.e., the AUC) within 180 min ( B ) of opsonized bacteria-induced (≥5 bacteria/cell) total ROS generated by 5 d calcitriol-differentiated THP-1 cells following preincubation with 80 ng/mL TNF for 48 h (i.e., added at day 3). Total ROS production was assessed via luminol-amplified chemiluminescence (in RLU/s) (representative experiment, n = 3). ( C , D ) Kinetics ( C ) and the respective cumulated production within 180 min ( D ) of opsonized bacteria-induced intracellular ROS generated by differentiated THP-1 cells following TNF preincubation as described in ( A ) (representative experiment, n = 3). ( E , F ) Kinetics ( E ) and the respective cumulated production within 30 min ( F ) of PMA-induced (100 nM) total ROS generated by primary human monocytes following TNF preincubation (80 ng/mL TNF for 48 h; representative experiment, n = 4). ( G ) Cumulated intracellular ROS production in MARCKS WT and KO clones (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3). ( H ) Cumulated intracellular ROS production in THP-1 and PKCβ KO cells (incubated as in ( A )) within 180 min following stimulation with bacteria (representative experiment, n = 3).

Article Snippet: For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP ® ), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP ® ), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP ® ; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Generated, Amplification, Clone Assay, Incubation