Journal: Frontiers in Cellular and Infection Microbiology

Article Title: PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis

doi: 10.3389/fcimb.2017.00231

Figure Lengend Snippet: PKC-η is involved in host MARCKS phosphorylation upon bacterial infection (A) A549 cells were infected with Bt CDC2721121 at MOI 50 or Y. enterocolitica WA at MOI 20. After the indicated times, cells were lysed and levels of phosphorylated Ser152/156 in the MARCKS effector domain (pMARCKS) were quantified using the PathScan ELISA kit and compared to uninfected A549 cells. Cells were also treated with 100 nM PMA as a positive control. In all samples, pMARCKS levels were normalized to the total MARCKS protein levels. The average and standard deviation from three independent experiments are shown. (B) A549 cells were treated with 25 nM siRNA against MARCKS, PKC-η, or non-targeting control (CTL) for 72 h prior to infection with Bt CDC2721121. At the indicated time, cells were lysed and the levels of pMARCKS were quantified as fold change relative to uninfected A549 cells. The average and standard deviation from three independent experiments are shown. The “ * ” denotes statistical significance ( p < 0.05) for fold pMARCKS change in samples treated with siRNA to MARCKS or PKC-η, compared to non-targeting siRNA samples (siR-CTL).

Article Snippet: Whole cell lysates were subjected to centrifugation at 12,000 g for 10 min at 4°C, and the resulting supernatants were analyzed using the PathScan Phospho-MARCKS (Ser152/156) Sandwich ELISA Kit (Cell Signaling Technology) following the manufacturer's instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Standard Deviation

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: (A) Biochemical PKC ADP ‐Glo kinase assay. Teleocidin A2 activates purified PKC mix containing isoforms α , β , and ɣ in high micromolar ranges in the presence of phosphatidylserine, but in the absence of diacylglycerol and calcium. Generated ADP during kinase reaction was measured using ADP ‐Glo ™ kinase assay. (B) PKC inhibitor Gö6983 reverses teleocidin A2‐mediated inhibition of PAR 2‐induced Ca 2+ release in MDA ‐ MB 231 cells. Inhibition of PKC with 1, 10, and 100 nmol/L Gö6983 has no impact on PAR 2‐induced intracellular Ca 2+ release. However, the inhibitory effect of 20 nmol/L teleocidin A2 on PAR 2 Ca 2+ release is reversed with increasing Gö6983 concentrations. Gö6983 was preincubated before teleocidin A2 addition and activation of PAR 2 with SLIGKV ‐ NH 2 . (C) PKC activation as detected by phospho‐ MARCKS (Ser152/156) ELISA ; f.i., fold increase. Data shown in bar chart are means ± SEM from three independent experiments performed in triplicates; ns, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001. (D) Inhibition of intracellular Ca 2+ mobilization by PKC activator PMA . Results represent means ± SEM , n ≥ 3 ( n , number of replicates). PKC, protein kinase C; PAR2, proteinase‐activated receptor 2; MDA‐MB 231, human breast adenocarcinoma cell line; PMA, phorbol 12‐myristate 13‐acetate.

Article Snippet: The PathScan ® phospho‐MARCKS (Ser152/156) sandwich ELISA (Cell Signaling Technology, Danvers, MA) was used to measure the level of cellular PKC activation in MDA‐MB 231 cells according to the manufacturer's information.

Techniques: Kinase Assay, Purification, Generated, Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Pharmacology Research & Perspectives

Article Title: Teleocidin A2 inhibits human proteinase‐activated receptor 2 signaling in tumor cells

doi: 10.1002/prp2.230

Figure Lengend Snippet: Effects of pan PKC inhibitor Gö6983, Rac1 inhibitor EH op‐016, and ROCK 1/2 inhibitor Y27632 on PAR 2‐mediated cell migration. (A) PKC inhibition does not significantly block PAR 2‐induced cell migration. (B) SLIGKV ‐ NH 2 induced intracellular PKC activation and inhibition by 100 nmol/L Gö6983 detected by phospho‐ MARCKS (Ser152/156) ELISA , f.i. fold increase. (C) Y27632 potentiates SLIGKV ‐ NH 2 ‐mediated cell migration. (D) SLIGKV ‐ NH 2 – PAR 2‐mediated cell migration is regulated via Rac1. Cell migration into detection zone was analyzed by particle analysis in ImageJ and basal migration after 48 h (control) was set to 100%. Data shown in bar chart are means ± SEM from three independent experiments with n ≥ 3 ( n , number of replicates); ns, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001. PKC, protein kinase C; Rac1, ras‐related C3 botulinum toxin substrate 1; ROCK1/2, Rho‐associated, coiled‐coil containing protein kinase 1/2; PAR, proteinase‐activated receptor; MARCKS , myristolated alanine‐rich protein kinase C substrate.

Article Snippet: The PathScan ® phospho‐MARCKS (Ser152/156) sandwich ELISA (Cell Signaling Technology, Danvers, MA) was used to measure the level of cellular PKC activation in MDA‐MB 231 cells according to the manufacturer's information.

Techniques: Migration, Inhibition, Blocking Assay, Activation Assay, Enzyme-linked Immunosorbent Assay