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

Journal: The Journal of Biological Chemistry

Article Title: LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells *

doi: 10.1074/jbc.M114.563742

Figure Lengend Snippet: Experiments demonstrating that LMTK2 localizes at the plasma membrane and co-immunoprecipitates with CFTR in human airway epithelial cells. A , schematic illustration of domain organization in CFTR and LMTK2. CFTR: TMD , transmembrane domain; NBD , nucleotide binding domain; R , regulatory domain. The R contains the phosphor-specific inhibitory Ser 737 site and other PKA consensus sites. LMTK2: TMD , transmembrane domain; KD , kinase domain. The residue Lys 168 located upstream of the Walker A motif is critical for kinase activity ( , ). MBD , myosin binding domain. Amino acid residues 567–773 mediate direct binding to myosin VI . TD , tail domain. B and C , immunoblots demonstrating similar distribution of LMTK2 at the plasma membrane of primary differentiated HBE cells and polarized human bronchial epithelial cell model CFBE41o- cells. The apical ( AP ) or basolateral ( BL ) plasma membrane ( PM ) proteins were isolated by domain-selective cell surface biotinylation in monolayers cultured on separate Transwell permeable growth supports. The whole cell lysate ( WCL ) and PM fraction of apically or basolaterally biotinylated monolayer were labeled AP and BL , respectively. Epithelial cell polarization is demonstrated by the basolateral localization of Na,K-ATPase. LMTK2 was detected at both membrane domains with AP:BL ratio of ∼1:1 when normalized for the corresponding LMTK2 abundance in WCL. WCL represents 5% of BT sample. D and E , immunoprecipitation experiments demonstrating that LMTK2 and CFTR co-immunoprecipitate in Calu-3 cells. CFTR was immunoprecipitated with the mouse monoclonal antibody M3A7 (IP CFTR, D ), and LMTK2 was immunoprecipitated with the rabbit polyclonal anti-LMTK2 antibody (IP LMTK2, E ). Mouse or rabbit non-immune IgGs were used as controls (IP IgG). WCL represents 2% of IP sample. Proteins were separated by SDS-PAGE using 7.5% gels and analyzed by immunobloting ( IB ) as indicated. All experiments were repeated three times from separate cultures with similar results.

Article Snippet: The following anti-human CFTR antibodies were used: mouse monoclonal, clone 596 (Cystic Fibrosis Foundation Therapeutics, Inc.; Chapel Hill, NC) and clone M3A7 (Millipore; Billerica, MA), and rabbit polyclonal Ab-737 (Assay Biotechnology Inc. San Francisco, CA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Residue, Activity Assay, Western Blot, Isolation, Cell Culture, Labeling, Immunoprecipitation, SDS Page