Journal: Journal of Natural Medicines

Article Title: Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl − /water secretion that involves cAMP-dependent activation of CFTR

doi: 10.1007/s11418-018-1207-9

Figure Lengend Snippet: Junchoto (JCT)-induced CFTR currents in HEK293T cells transiently transfected with CFTR. a Representative records of whole-cell current activation in CFTR- ( left ) and vector-transfected ( right ) cells before and after application of 400 μg/mL JCT ( filled bars ), taken during the application of alternating pulses from 0 to ± 40 mV every 10 s. The asterisks denote times when step pulses were applied. The inset shows a membrane displaying immunoblot of CFTR protein from control (vector-transfected) and CFTR transfected HEK293T cells in the upper lane. Note that the lower band is only detected in CFTR-transfected HEK293T cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels in the lower lane. b The current response to step pulses from −100 to +100 mV for CFTR ( top traces ) and the vector ( bottom trace ). c I – V relationships for the mean JCT-activated current densities for cells expressing CFTR ( open circles ; n = 6) and vector ( filled triangles ; n = 6)

Article Snippet: The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA).

Techniques: Transfection, Activation Assay, Plasmid Preparation, Membrane, Western Blot, Control, Expressing

Journal: Journal of Natural Medicines

Article Title: Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl − /water secretion that involves cAMP-dependent activation of CFTR

doi: 10.1007/s11418-018-1207-9

Figure Lengend Snippet: Effects of small interfering RNA (siRNA) silencing of CFTR on JCT-induced anion currents in Caco-2 cells. a RT-PCR analysis of CFTR mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in control, mock-treated and CFTR siRNA-treated Caco-2 cells. The data represent 3 similar experiments. No PCR product was amplified when reverse transcriptase was omitted from the reaction in the RT(−) group. The nucleotide sequences of the PCR products obtained with CFTR-specific primers were completely identical to the corresponding sequences in human CFTR (4302-4722 Sequence ID: NM_000492.3). b Immunoblot of CFTR protein from control, mock-treated and CFTR siRNA-treated Caco-2 cells. Alpha-tubulin bands with molecular mass 50 kDa were detected at equal levels. c – h Representative time courses of the JCT-evoked whole-cell currents recorded at +100 and −100 mV under ramp clamp in mock siRNA-treated cells ( c ) and in CFTR siRNA-treated cells ( e ). Gray bar and solid bar show application of 400 μg/mL of JCT and 10 μM of FSK, respectively. Corresponding I – V relationships at time points a–c in d and a–c in f . g , h Averages of JCT-induced whole-cell current at +100 mV in mock and CFTR siRNA ( g ), and FSK-induced whole-cell current ( h ) ( n = 5–6). Data points show the mean ± SEM. * P < 0.05 compared to mock at +100 mV

Article Snippet: The blots were incubated with anti-CFTR antibody (1:1000 dilution, CUSABIO and CUSAb, MD, USA: CSB-PA001608) or monoclonal anti-α-tubulin (as an internal standard, 1:2000 dilution; Sigma-Aldrich: T6074), and stained using the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL, USA).

Techniques: Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Control, Amplification, Reverse Transcription, Sequencing, Western Blot