Journal:

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

doi:

Figure Lengend Snippet: Yeast two-hybrid interaction maps of α1-AMPK and CFTR COOH-terminal tail. (a) Determination of regions of α1-AMPK important for interaction with CFTR-1411–1480. Functional regions of α1-AMPK are shown schematically at the top, as estimated by sequence homology to yeast Snf1p (38). Constructs containing residues upstream of amino acid 161 were made by high-fidelity PCR amplification using appropriate primers and full-length rat α1-AMPK cDNA (31) as template. Other constructs were either original prey clones identified in the two-hybrid screen (α1-161-550 and 294-550) or were generated by PCR using α1-294-550 prey plasmid as a template. (b) Determination of region and specific residues within CFTR-1411–1480 important for interaction with α1-294-550. Expression of the CFTR-1420–1443 fragment could not be detected by Western analysis. Because lack of interaction observed may have resulted from no expression, it is uncertain whether residues 1444–1457 are required for strong interaction.

Article Snippet: Beads were washed with lysis buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary rabbit polyclonal antibodies to α1-AMPK (1:1000 dilution), prepared as described previously ( 31 ), followed by secondary anti-rabbit IgG-HRP antibodies (1:10,000 dilution; Boehringer-Mannheim, Indianapolis, Indiana, USA).

Techniques: Functional Assay, Sequencing, Construct, Amplification, Clone Assay, Two Hybrid Screening, Generated, Plasmid Preparation, Expressing, Western Blot

Journal:

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

doi:

Figure Lengend Snippet: Confirmation of AMPK-CFTR interaction by GST pull-down assays. (a) CFTR COOH-terminal tail (GST-CFTR-1411–1480) pulls down α1-AMPK in vitro. Purified liver AMPK holoenzyme (15 ng) was loaded in first lane as reference. (b) GST-α1-AMPK fusion protein expressed in CHO-BQ2 cells pulls down full-length CFTR in vivo. Ten micrograms of the total soluble cellular protein was loaded in first and third (lysate) lanes as reference for CFTR. Second and fourth (beads) lanes show eluate from GSH-Sepharose beads after affinity purification and washing. (c) Comparison of binding strengths of CFTR with NH2-terminal and COOH-terminal GST-α1-AMPK fusion proteins in CHO-BQ2 cells. Lower panel is the same membrane probed with anti-GST antibodies to allow comparisons of different CFTR bands in panel above. (d) GST-α2-AMPK fusion protein expressed in CHO-BQ2 cells pulls down CFTR in vivo. All results shown are representative of at least three replicate experiments.

Article Snippet: Beads were washed with lysis buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary rabbit polyclonal antibodies to α1-AMPK (1:1000 dilution), prepared as described previously ( 31 ), followed by secondary anti-rabbit IgG-HRP antibodies (1:10,000 dilution; Boehringer-Mannheim, Indianapolis, Indiana, USA).

Techniques: In Vitro, Purification, In Vivo, Affinity Purification, Binding Assay

Journal:

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

doi:

Figure Lengend Snippet: Western blot of lysates from various cell lines. Total protein (10–20 μg per lane) was loaded on a 10% gel and immunoblotted with anti-α1-AMPK antibody (1:1,000), followed by anti-rabbit IgG-HRP (1:10,000).

Article Snippet: Beads were washed with lysis buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary rabbit polyclonal antibodies to α1-AMPK (1:1000 dilution), prepared as described previously ( 31 ), followed by secondary anti-rabbit IgG-HRP antibodies (1:10,000 dilution; Boehringer-Mannheim, Indianapolis, Indiana, USA).

Techniques: Western Blot

Journal:

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

doi:

Figure Lengend Snippet: Immunohistochemistry of tissue sections from rat nasal mucosa. Contiguous sections were stained using (a) anti-CFTR or (b) anti-α1-AMPK antibodies, followed by biotinylated secondary antibodies and rhodamine-avidin. Both sections are also DAPI-nuclear stained (blue). Bar, 100 μm. Additional contiguous sections stained without addition of primary antibody revealed little nonspecific staining (not shown).

Article Snippet: Beads were washed with lysis buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary rabbit polyclonal antibodies to α1-AMPK (1:1000 dilution), prepared as described previously ( 31 ), followed by secondary anti-rabbit IgG-HRP antibodies (1:10,000 dilution; Boehringer-Mannheim, Indianapolis, Indiana, USA).

Techniques: Immunohistochemistry, Staining, Avidin-Biotin Assay

Journal:

Article Title: Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

doi:

Figure Lengend Snippet: In vitro phosphorylation of CFTR by AMPK. CFTR was immunoprecipitated from CHO-BQ2 cell lysates (lanes 2–5) before phosphorylation, SDS-PAGE, and autoradiography. In lane 2, no exogenous kinase was added to the phosphorylation buffer. In lanes 3 and 4, affinity-purified AMPK holoenzyme was added to immunoprecipitated CFTR in the absence or presence of 0.2 mM AMP, respectively. Affinity-purified α1-1-312 was added to immunoprecipitate in lane 5. As a negative control, AMPK holoenzyme was added to an immunoprecipitate from CHO cells that do not express CFTR (lane 1). Results shown are representative of three replicate experiments.

Article Snippet: Beads were washed with lysis buffer, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary rabbit polyclonal antibodies to α1-AMPK (1:1000 dilution), prepared as described previously ( 31 ), followed by secondary anti-rabbit IgG-HRP antibodies (1:10,000 dilution; Boehringer-Mannheim, Indianapolis, Indiana, USA).

Techniques: In Vitro, Immunoprecipitation, SDS Page, Autoradiography, Affinity Purification, Negative Control

Journal: Diagnostics

Article Title: A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

doi: 10.3390/diagnostics12030561

Figure Lengend Snippet: MyoProfiler: detection and segmentation of myofibers using anti-laminin, α1-stained muscle cross-sections. ( A ) Workflow of the first part of MyoProfiler pipeline. ( B ) RescaleIntensity module for laminin signal. Scale bar = 100 µm. ( C ) Custom “unsharp mask” step (GaussianFilter and ImageMath 1+2) for sharpening myofiber boundaries. ( D ) Line structures and myofiber boundaries are further enhanced with 4 sequential modules (two EnhanceAndSuppressFeatures modules and two ImageMath modules). ( E ) Closing module closes the intensity gaps between pixels. ( F ) MedianFilter module reduces salt-and-pepper background noise. ( G ) Threshold module detects positive signal and produces a binary image. ( H ) Morph module closes the gaps between muscle fibers and fills small holes. ( I ) Inverted binary image (ImageMath 5). ( J ) Color map image of segmented myofibers with IdentifyPrimaryObjects module. FilterObjects module discards wrong muscle fibers (magenta arrows point to discarded fibers). ( K ) OverlayOutlined generated an output image in which both segmented (red) and discarded (yellow outlines and pointed by cyan arrows) myofibers are outlined.

Article Snippet: Primary antibodies used for this study were rabbit polyclonal antibody raised against laminin, α1 (Sigma-Aldrich, Merck KGaA, Burlington, MA, USA; Cat#: L9393, RRID:AB_477163, 1:500) and rat monoclonal antibody raised against F4/80 (Bio-Rad Laboratories, Hercules, CA, USA; Cat#: MCA497G, RRID:AB_872005, 1:300).

Techniques: Staining, Generated

Journal: Diagnostics

Article Title: A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

doi: 10.3390/diagnostics12030561

Figure Lengend Snippet: Quantitative measurements automatically performed with MyoProfiler and compared with Fiji software. ( A ) Representative immunofluorescence images of tibialis anterior sections from wild-type (WT) and mdx mice. Sections were stained with anti-laminin, α1 (green) and anti-F4/80 (red) antibodies and counterstained with DAPI (blue). Scale bar = 50 µm. ( B ) Minimum Feret diameter (MFD) quantification and fiber size distribution of myofibers. ( C ) Quantification of the number of nuclei. ( D ) Quantification of centrally nucleated (CNFs) and peripherally nucleated fibers (PNF). ( E ) Quantification of the number of nuclei per CNF performed by CellProfiler. ( F ) Quantification of the percentage of macrophages. Data are expressed as mean ± SEM, and unpaired t -test was used for comparison (N = 3 for WT and N = 4 for mdx ; * = p < 0.05; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Primary antibodies used for this study were rabbit polyclonal antibody raised against laminin, α1 (Sigma-Aldrich, Merck KGaA, Burlington, MA, USA; Cat#: L9393, RRID:AB_477163, 1:500) and rat monoclonal antibody raised against F4/80 (Bio-Rad Laboratories, Hercules, CA, USA; Cat#: MCA497G, RRID:AB_872005, 1:300).

Techniques: Software, Immunofluorescence, Staining, Comparison