rabbit anti inca antibody  (Cell Signaling Technology Inc)

Journal: Infection and Immunity

Article Title: Host and Bacterial Glycolysis during Chlamydia trachomatis Infection

doi: 10.1128/IAI.00545-20

Figure Lengend Snippet: F-actin, host cell microtubule dynamics, and vesicular trafficking do not play a role in the localization of aldolase A at the inclusion. HeLa cells expressing 3×FLAG-AldoA and infected with the mCherry CtL2 strain were fixed at 24 h postinfection and immunostained with anti-FLAG antibodies. (A, D, and E) Quantification of the percentage of inclusions positive for 3×FLAG-AldoA following treatment with 1 μM cytochalasin D (Cyto D) at 23.5 h postinfection (A), 33 μM nocodazole at 23.5 h postinfection (D), or 1 μg/ml brefeldin A at 6 h postinfection (E). (B) Quantification of the percentage of inclusions positive for 3×FLAG- AldoA in HeLa cells expressing wild-type 3×FLAG-AldoA (WT), or point mutants no longer able to bind actin 3×FLAG-R42A AldoA (R42A) and 3×FLAG-R148A AldoA (R148A). (C) Quantification of the percentage of inclusions positive for 3×FLAG-AldoA in HeLa cells infected with wild-type C. trachomatis (WT) or an inaC mutant strain (inaC::aadA). Data show the means and the SD of a combination of three independent experiments. ns, not statistically significant (Student t test).

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy (IF) and immunoblotting (Western blotting): mouse monoclonal anti-FLAG (1:1,000 for IF; Sigma), rabbit polyclonal anti- C. trachomatis IncA (1:200 for IF; kindly provided by T. Hackstadt, Rocky Mountain Laboratories), rabbit polyclonal anti-HA (1:100 for IF; Sigma), mouse monoclonal anti-aldolase A (1:1,000 for Western blotting; Santa Cruz), rabbit polyclonal anti-actin (1:10,000 for Western blotting; Sigma), mouse monoclonal anti-α-tubulin (1:2000 for IF; Sigma), and Alexa Fluor 514-conjugated phalloidin (1:200 for IF; Invitrogen).

Techniques: Expressing, Infection, Mutagenesis

Journal: Infection and Immunity

Article Title: Host and Bacterial Glycolysis during Chlamydia trachomatis Infection

doi: 10.1128/IAI.00545-20

Figure Lengend Snippet: Aldolase A confers a developmental advantage to C. trachomatis. (A) HeLa cells expressing 3×FLAG-tagged AldoA were infected with the mCherry CtL2 strain and the area of 3×FLAG-AldoA positive and negative inclusions (arbitrary units; each circle represents data from a single inclusion) was quantified at 24 h postinfection. (B to D) HeLa cells were transfected with individual aldolase A siRNA duplexes or siRNA buffer alone (Mock) for 3 days. (B) Immunoblots of the corresponding lysates were probed using antibodies against endogenous aldolase A (top blot) and actin (bottom blot). The knockdown efficacy of each duplex targeting aldolase A is indicated. (C and D) HeLa cells treated with the indicated siRNA duplexes targeting aldolase A were infected with C. trachomatis. For each condition, the total area of the inclusions (arbitrary units; each circle represents data from a single inclusion) at 32 h postinfection (C) and the number of infectious bacteria (IFUs/ml) at 48 h postinfection (D) was determined. The cell density of the mock-treated conditions was matched to the cell density of the siRNA-treated conditions. The data in panel A show the mean and the standard errors of the mean (SEM) of a representative experiment. The data in panel C show the means and the SEM of a combination of three independent experiments. The data shown in panel D is from a representative experiment. Error bars indicate the SD. ****, P < 0.0001 (Student t test).

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy (IF) and immunoblotting (Western blotting): mouse monoclonal anti-FLAG (1:1,000 for IF; Sigma), rabbit polyclonal anti- C. trachomatis IncA (1:200 for IF; kindly provided by T. Hackstadt, Rocky Mountain Laboratories), rabbit polyclonal anti-HA (1:100 for IF; Sigma), mouse monoclonal anti-aldolase A (1:1,000 for Western blotting; Santa Cruz), rabbit polyclonal anti-actin (1:10,000 for Western blotting; Sigma), mouse monoclonal anti-α-tubulin (1:2000 for IF; Sigma), and Alexa Fluor 514-conjugated phalloidin (1:200 for IF; Invitrogen).

Techniques: Expressing, Infection, Transfection, Western Blot, Knockdown, Bacteria

Journal: Infection and Immunity

Article Title: Host and Bacterial Glycolysis during Chlamydia trachomatis Infection

doi: 10.1128/IAI.00545-20

Figure Lengend Snippet: Chlamydia glycolytic enzymes are downregulated during development. The relative gene expression of a subset of C. trachomatis glycolytic enzymes (glucose-6-phosphate isomerase, pgi; aldolase A, dhnA; pyruvate kinase, pykF) at 12, 24, 36, and 48 h postinfection of HeLa cells was determined by RT-qPCR. Gene expression was normalized to 16S rRNA levels and the expression at 8 h postinfection (dotted line). The late-expressed gene encoding the outer membrane protein OmcA (omcA) was included as a control. The y axis is a log scale. The data show the means and the SD of a combination of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student t test).

Article Snippet: The following primary antibodies were used for immunofluorescence microscopy (IF) and immunoblotting (Western blotting): mouse monoclonal anti-FLAG (1:1,000 for IF; Sigma), rabbit polyclonal anti- C. trachomatis IncA (1:200 for IF; kindly provided by T. Hackstadt, Rocky Mountain Laboratories), rabbit polyclonal anti-HA (1:100 for IF; Sigma), mouse monoclonal anti-aldolase A (1:1,000 for Western blotting; Santa Cruz), rabbit polyclonal anti-actin (1:10,000 for Western blotting; Sigma), mouse monoclonal anti-α-tubulin (1:2000 for IF; Sigma), and Alexa Fluor 514-conjugated phalloidin (1:200 for IF; Invitrogen).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Membrane, Control

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