Journal: medRxiv

Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy

doi: 10.1101/2024.08.17.24311658

Figure Lengend Snippet: (A) Urinary β2-microglobulin level of individuals with biallelic pathogenic mutations in VPS35L or CCDC93 . (B) The schematic illustration depicts the chimeric constructs of Human-LRP2 utilized in the study. In addition to the wild-type cytoplasmic sequence, mutant constructs were generated in which either or both NPxY motifs were substituted with NPxA. (C) Representative blots of mCherry-nanotrap for mCherry-SNX17 under co-overexpression of chimeric constructs of LRP2. GFP-tagged cytoplasmic tail of CI-MPR, a SNX-BAR cargo protein, was used as negative control. Bar graphs show band intensities relative to WT calculated from three independent experiments. (D) Representative blots for LRP1, LRP2, and N-Cadherin from three independent experiments. Cell surface protein fractions were obtained from HEK293T cell lines. Bar graphs show relative values of KO cells to their rescue or parental cells. (E) Representative view of mCherry-D3 uptake in parental, VPS35L-KO, and VPS35L-rescue cells. Cells were incubated with mCherry-D3 for 30 min, followed by DAPI staining and imaging with a fluorescence microscope. mCherry intensity was quantified using Image J software. 10 fields were acquired in each condition in each of three independent experiments, and mCherry intensity of each field was normalized to the number of DAPI-stained nuclei. Scale bars, 10 µm. (F) HEK293T cell lines were incubated with mCherry-D3 for 30 min followed by FACS analysis to quantitate cellular uptake of mCherry-D3. (G) Immunohistochemistry of Lrp2 in renal tissue in littermate control or Vps35l-cKO Nestin . Red arrows indicate Lrp2 in S1 segment of proximal tubules. Three mice were analyzed in each group. G; Glomerulus. (H) Schematic illustration of the molecular mechanism underlying the proteinuria observed in Ritscher-Schinzel syndrome. (C, D, E) Error bars represent mean± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).

Techniques: Construct, Sequencing, Mutagenesis, Generated, Over Expression, Negative Control, Incubation, Staining, Imaging, Fluorescence, Microscopy, Software, Immunohistochemistry, Control

Journal: Scientific Reports

Article Title: Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

doi: 10.1038/s41598-024-58234-0

Figure Lengend Snippet: ( A ) Neighbor-joining phylogenic tree of amino acid sequences encoding Kir channel subunits in insects and humans. Geneious Prime 2023.2.2 ( www.geneious.com ) was used to construct the tree. The Genbank® accession numbers of the Kir sequences are shown in parentheses. Underlines denote Apis mellifera Kir channel subunits. The abbreviations of the species are as follows: Aag: Aedes aegypti , Agam: Anopheles gambiae , Agly: Aphis glycines , Amel: Apis mellifera , Apisu: Acyrthosiphon pisum , Clec: Cimex lectularius , Dmel: Drosophila melanogaster , Hsap: Homo sapiens , Nlug: Nilaparvata lugens . ( B ) Evaluation of expression of AmKir channel isotypes in honeybee tissues and life stage. Tissue-specific expression of the AmKir channel isotype was determined using RT-PCR. All honeybee tissue samples underwent identical preparation steps, from dissection to gel electrophoresis. The sizes of the ladder marker next to the fist blot is the same for all the other blots, and are indicated in base pairs (bp). Abbreviations: LE: legs, LA: larvae, HE: heads, BR: brains, GAG: ganglia, GU: guts, MU: muscles, ANT: antennae ( C ) Sequence alignments of the three current-generating AmKir channel isoforms were performed using MegAlign from Lasergen. The blue squares indicate the hydropathy estimation of the transmembrane regions. The red square represents the estimated region of the selectivity filter. The black squares indicate arginine-glutamate salt bridge interactions forming the PIP 2 -binding site in the cytoplasmic domain.

Article Snippet: Abbreviations: LE: legs, LA: larvae, HE: heads, BR: brains, GAG: ganglia, GU: guts, MU: muscles, ANT: antennae ( C ) Sequence alignments of the three current-generating AmKir channel isoforms were performed using MegAlign from Lasergen.

Techniques: Construct, Expressing, Reverse Transcription Polymerase Chain Reaction, Dissection, Nucleic Acid Electrophoresis, Marker, Muscles, Sequencing, Binding Assay

Journal:

Article Title: NHERF family and NHE3 regulation

doi: 10.1113/jphysiol.2005.090399

Figure Lengend Snippet: PDZ domains of the NHERF family were defined using ScanSite MotifScan. The 12 human NHERF family PDZ domains were individually analysed for sequence relationship by the MegAlign program (DynaStar, Inc.). The domains were grouped by closest relationships. Relationship with C. elegans and D. melanogaster proteins were also analysed by MegAlign. All protein names and sequences were obtained from NCB1. C. elegans has two PDZ domain-containing proteins that are related to the NHERF family. CO1F6.6 contains one PDZ domain while 4J893 contains two PDZ domains, one identical to that in CO1F6.6 and the other 5% identical. This suggests that the second PDZ domain of 4J893 may have evolved by gene duplication. Each of the two worm PDZ domains is a precursor to certain groups of PDZ domains of the human NHERF family. The D. melanogaster proteins CG10939-PA and CG32758-PA each contain one PDZ domain (10% identical to each other) and are precursors to the human NHERF family.

Article Snippet: The 12 human NHERF family PDZ domains were individually analysed for sequence relationship by the MegAlign program (DynaStar, Inc.).

Techniques: Sequencing