camp analog 8 cpt camp  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: The EPAC–Rap1 pathway prevents and reverses cytokine-induced retinal vascular permeability

doi: 10.1074/jbc.M117.815381

Figure Lengend Snippet: EPAC contributes to basal barrier properties in BREC. A, solute flux assay was used to test EPAC antagonist, HJC0350, at different concentrations on BREC. HJC0350 5 to 10 μm had a significant increase in basal permeability and blocked 8-CPT-AM barrier induction. Average Po values for control and HJC0350 were 2.2 × 10−7 and 4.4 × 10−7 (cm/s). Results are expressed as the mean ± S.D. relative to the control with a total of n ≥ 3, ####, p < 0.0001 or ##, p < 0.001 compared with control or scramble control in B and C and ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05 compared with 8-CPT-AM or siRNA EPAC1 in B or siRNA EPAC1 and EPAC2 in C. B, solute flux assay was used to test permeability in BREC with either EPAC1 or EPAC2 knockdown. Scramble (ctrl), EPAC1 and EPAC2 siRNA at 100 nm were used in BREC. Average Po values for scramble control, EPAC1 and EPAC2 siRNA were 1.1 × 10−7, 3.1 × 10−7, and 1.3 × 10−7 (cm/s). C, solute flux assay was used to test permeability in BREC with double knockdown of EPAC1 and EPAC2 in BREC. EPAC1 and EPAC2 siRNA were used at 100 nm. Average Po values for scramble control and double EPAC1 and 2 siRNA were 0.86 × 10−6 and 3.1 × 10−6 (cm/s). D, SYBR Green qRT-PCR was used to quantify EPAC1 and E, EPAC2 mRNA after siRNA knockdown.

Article Snippet: The following reagents were purchased from Tocris Bioscience: the cAMP analog 8-CPT-AM specific for EPAC (catalogue number 4853), EPAC2 inhibitor (HJC 0350) 2,4-dimethyl-1-[(2,4,6-trimethylphenyl) sulfonyl]-1H-pyrrole (catalogue number 4844), and EPAC inhibitor (ESI 09) α-[(2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-β-oxo-3-isoxazolepropanenitrile (catalogue number 4773).

Techniques: Flux Assay, Permeability, Control, Knockdown, SYBR Green Assay, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: The EPAC–Rap1 pathway prevents and reverses cytokine-induced retinal vascular permeability

doi: 10.1074/jbc.M117.815381

Figure Lengend Snippet: Rap1B contributes to basal permeability and tight junction organization. A, both Rap1 (Rap1A and Rap1B) and EPAC (EPAC1 and EPAC2) isoforms were found to be present in BREC by PCR. B, SYBR Green qRT-PCR was used to test Rap1 isoform siRNAs specificity. #, p < 0.05. C, Western blot shows that composite Rap1B (100 nm) siRNA results in a 66% knockdown, Student's t test ####, p < 0.0001, n ≥ 9. D, solute flux assay was used to test permeability in BREC with Rap1B knockdown. Scramble and Rap1B siRNA at 100 nm were used in BREC. Average Po values for scramble control and Rap1B siRNA control were 2.4 × 10−7 and 5.4 × 10−7 (cm/s). E, solute flux assay was used to test permeability in BREC with individual Rap1B-1 siRNA. Scramble and Rap1B-1 siRNA at 100 nm were used in BREC. Average Po values for scramble control and Rap1B-1 siRNA control were 9.0 × 10−8 and 2.3 × 10−7 (cm/s). F, immunofluorescence staining of TJ proteins ZO-1, occludin, and claudin-5 was performed to assess the organization of TJ proteins after Rap1B knockdown with and without VEGF (50 ng/ml) for 1 h. Scale bar, 10 μm. Histograms of scoring TJ show % organization of TJs. All results are expressed as the mean relative to the scramble control with a total of n > 4. ZO-1, occludin, and claudin-5 border staining were assessed by semi-quantitative ranking score system based on scale grades from 1 to 5. Immunofluorescence results are expressed as the mean relative to the scramble control n ≥ 4 with analysis by one-way analysis of variance and Bonferroni post hoc test, ****, p < 0.0001 compared with scramble control and ##, p < 0.05; ###, p < 0.001; ####, p < 0.0001 compared with Rap1B siRNA VEGF. Permeability results are expressed as the mean relative to the scramble control two-way analysis of variance analysis and Bonferroni post hoc test: *, p < 0.05; **, p < 0.01; and ##, p < 0.01 comparison between scramble and Rap1B or Rap1B-1 siRNA controls.

Article Snippet: The following reagents were purchased from Tocris Bioscience: the cAMP analog 8-CPT-AM specific for EPAC (catalogue number 4853), EPAC2 inhibitor (HJC 0350) 2,4-dimethyl-1-[(2,4,6-trimethylphenyl) sulfonyl]-1H-pyrrole (catalogue number 4844), and EPAC inhibitor (ESI 09) α-[(2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-β-oxo-3-isoxazolepropanenitrile (catalogue number 4773).

Techniques: Permeability, SYBR Green Assay, Quantitative RT-PCR, Western Blot, Knockdown, Flux Assay, Control, Immunofluorescence, Staining, Comparison