PMC

Raf-1 regulation by cAMP involves multiple phosphorylation events. (A) mRaf-1 kinase activity. mRaf-1 (Raf), m^A233Raf-1 (A233), m^A43,A233Raf-1 (A43, A233), m^A233,A259Raf-1 (A233, A259) or m^{A43,A233,A259}Raf-1 (A43,A233,A259) were transiently expressed in COS cells and treated as for Fig. 2B for the Raf-1 kinase assay. The results are for one experiment assayed in triplicate, with error bars indicating standard deviations from the mean. Similar results were obtained in two independent experiments. (B) S43 phosphorylation. mRaf-1 (Raf), m^A233Raf-1 (A233), m^A259Raf-1 (A259), m^A233,A259Raf-1 (A233,A259) or m^{A43,A233,A259}Raf-1 (A43,A233,A259) were transiently expressed in COS cells which were either untreated or treated for 10 min with forskolin and IBMX as indicated. An equal amount of myc-tagged Raf-1 protein was immunoprecipitated and probed with a phospho-specific antibody to S43. Similar results were obtained in two independent experiments.

Using CLUSTAL W software aligned amino acid sequences of the Streptomyces coelicolor A3(2), Neisseria gonorrhoeae, E. coli, and D. radiodurans. Identical amino acids are highlighted in black, and conserved residues are highlighted with grey. The DrOxyR helix-turn-helix region has four conserved residues (R4, L32, S33 and R50) [17]. At its LysR-substrate binding domain, D142 and R273 possibly define an activating region on OxyR (contact with RNA polymerase)[18], A233 residue is involved in tetramerization[17], V110, L124, and A233 form a hydrophobic core[58]. Numbering is based on the E. coli OxyR sequence.

Properdin augments AP-mediated C3 deposition on N. gonorrhoeae. A. C3 deposition on 5 strains of N. gonorrhoeae incubated with NHS-Mg/EGTA (20% (v/v) alone (grey shaded histograms) or with added anti-P mAb (Quidel mAb A233) that blocks properdin function (histograms depicted by solid lines). B. C3 deposition on gonococcal strains after incubation with P-depleted serum-Mg/EGTA (20% (v/v)) (histograms depicted by solid lines) or with P-depleted serum-Mg/EGTA reconstituted with purified unfractionated P (grey shaded histograms). Numbers adjacent to histograms represent the median fluorescence intensities (MFIs) of C3 binding. Detection of C3 fragments was performed as in Fig. 1A. One representative experiment of two separately performed and reproducibly repeated experiments is shown for each of the histograms..

Positions of constitutive and activation-defective mutations on the OxyR structure. Space-filling model of the OxyR regulatory domain in the reduced (A) and oxidized (B) conformations was generated by using InsightII software (Accelrys Software, Inc.). The front view is given on the left, and the back view is on the right. One monomer is shown in aqua, and the second monomer is shown in green. The positions of the C199 and C208 residues are indicated in yellow. The residues affected by the constitutive mutations (T100, H114, H198, R201, and A233) isolated in a previous screen for elevated expression of an oxyS-galK fusion (8) are indicated in red. The residues found to be mutated in the activation-defective mutants (G96, G102, P103, H125, E126, T129, D142, F219, T238, and R273) described here are indicated in orange. The black box surrounds residues D142 and R273.