Journal: EBioMedicine

Article Title: Chlamydia trachomatis Prevents Apoptosis Via Activation of PDPK1-MYC and Enhanced Mitochondrial Binding of Hexokinase II

doi: 10.1016/j.ebiom.2017.08.005

Figure Lengend Snippet: CTL2 induces PDPK1 phosphorylation downstream of PI3K. a) Western blot showing an increase of PDPK1 phosphorylated at Ser-241 compared to total PDPK1 at 24 and 48 h p.i. in CTL2-infected (MOI 1) whole cell lysates. β-actin served as loading control. b) The band densities from a) were quantified and normalized to corresponding band densities of the β-actin loading control. Alterations in expression levels compared to non-infected controls are represented as mean fold change ± SEM, * p < 0.05, t -test, n = 3. c) CTL2 infection increases levels of PDPK1 phosphorylated at Ser-241, which is recruited as a rim-like structure at the inclusions in HeLa cells. PDPK1 phosphorylated at Ser-241, IncA and nuclei were labelled with Cy3-conjugated phosphorylated PDPK1 and IncA antibodies and DAPI. Scale bar: 30 μm. Asterisks are CTL2 inclusions. d) Monolayers of fallopian tube mesenchymal stem cells, infected with CTL2 for 48 h p.i. (MOI 0.5) were labelled with antibodies against Chlamydia trachomatis , PDPK1 phosphorylated at Ser-241 and DAPI. Results were similar to c). Scale bar: 30 μm. e) Human primary fallopian tube epithelial organoids, infected with CTL2 for 48 h p.i. were labelled with antibodies against Chlamydia trachomatis , PDPK1 phosphorylated at Ser-241 and E -Cadherin. Results were similar to c). Scale bar: 30 μm.

Article Snippet: The following antibodies were used: rabbit monoclonal antibodies against total MYC (D84C12, 5605), total HKII (C64G5, 2867) and total PDHK1 (C47H1, 3820), rabbit polyclonal antibodies against total PDPK1 (3062) and anti-P-PDPK1 (Ser-241, 3061) (all Cell Signaling); rabbit polyclonal anti-P-MYC (T58, ab28842), mouse monoclonal anti-P-MYC (S62, 33A12E10, ab78318, both from Abcam); mouse monoclonal anti-VDAC1 (B-6, sc-390996), Rho A (24C4, sc-418) and anti-MYC (9E10, sc-40 all from Santa Cruz Biotechnology Inc.); mouse monoclonal anti- Chlamydia trachomatis MAb species-specific KK-12 IgG2a (supplied by David Grayston, University of Washington, Seattle); rabbit polyclonal anti- Chlamydia genus-specific antibody (AG, 3-090, Milan Analytica), mouse monoclonal anti- Chlamydia trachomatis Hsp60 (ALX-804-072-R100, Enzo Life Sciences), rabbit anti-INCA antibody ( ); goat anti-chlamydia (1990-0404, AbD Serotec); rabbit monoclonal cleaved PARP (Asp214) and rabbit polyclonal anti-cleaved caspase 3 (Asp175, 9661, both from Cell Signaling).

Techniques: Phospho-proteomics, Western Blot, Infection, Control, Expressing

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells were infected with Ctr- EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as . (B) HeLa cells were infected with Ctr- EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or Ctr -infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course Ctr- infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with Ctr -pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.

Article Snippet: Polyclonal serum against IncA (anti-rabbit) was obtained through Gramsch laboratories.

Techniques: Infection, Homogenization, Clinical Proteomics, Membrane, Isolation, Activation Assay, Control, Marker, Transfection, Staining