Journal: Cell reports

Article Title: TRPM2 enhances ischemic excitotoxicity by associating with PKCγ.

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: Figure 6. TAT-M2PBM protects mice against ischemic stroke (A) Co-immunoprecipitation of PKCg, GluN2a, and GluN2b by TRPM2 in the brain from WT mice 2, 12, and 24 h after injection with TAT-SC (scramble) or TAT- M2PBM at 100 nmol/kg. (B) Injection strategy for evaluating short-term protective effects. (C and D) Brain injury in WT and Trpm2 deletion (M2KO) mice injected with TAT-SC or TAT-M2PBM (100 nmol/kg) 24 h after MCAO (n = 8, 7, 5, 5). (C) Triphenyl tetrazolium chloride (TTC) staining showing infract area (white). (D) Quantification of brain infarction and neurological deficit score. (E and F) Brain injury in WT mice injected with TAT-SC, TAT-EE3, or TAT-M2PBM 24 h after MCAO (n = 7, 9, 9). (E) TTC staining showing infract area (white). (F) Quantification of brain infarction and neurological deficit score. (G) Injection strategy for evaluating short-term protective effects. (H–K) Brain injury in WT and M2KO mice injected with TAT-SC or TAT-M2PBM 7 days after MCAO (n = 8, 9). (H) TTC staining showing infract area (white). (I and J) Quantification of brain infarction and neurological deficit score. (K) Quantification of rotarod test. ns, no statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001; unpaired t test; mean ± SEM; scale bar: 5 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Pierce Cell Surface Protein Isolation Kit Thermal Fisher Scientific 89881 ProteoExtractTM Native Membrane Protein Extraction Kit Calbiochem 444810 Experimental models: Cell lines HEK293T cells ATCC CRL-3216 Experimental models: Organisms/strains C57BJ6 mice JAX 000664 Global TRPM2 knockout mice Yasuo Mori’s lab at Kyoto University N/A Oligonucleotides Primers for mutagenesis and subcloning; see Table S1 This study N/A Recombinant DNA GluN1A Luo et al.40 Addgene, 17928 GluN2A Luo et al.40 Addgene, 17924 GluN2B Luo et al.40 Addgene, 17925 PKC-g Oancea et al.41 Addgene, 112266 PKC-g-DN Soh et al.42 Addgene, 21239 CKAR Violin et al.17 Addgene, 14860 pET15b vector containing a removable TEV protease recognition site Zong et al.6 MilliporeSigma 69661–3 pGEX-4T3 vector containing a removable tobacco etch virus (TEV) protease recognition site Zong et al.6 NovoPro V010916 pcDNA4/TO-FLAG-hTRPM2 Sharenberg AM at University of Washington N/A Software and algorithms Prism 9 Graphpad https://www.graphpad.com/ Photoshop 2020 Adobe https://www.adobe.com/ Illustrator 2020 Adobe https://www.adobe.com/ NIS-Elements Nikon N/A ImageJ Schneider et al.43 https://imagej.net/ij/ Other Rotarod Maze https://maze.conductscience.com Avestin EmulsiFlex C3 ATA Scientific Instruments https://www.atascientific.com.au/ products/avestin-emulsiflex-c3 Glutathione Sepharose 4B column GE Healthcare Discontinued Ni2+-nitrilotriacetic acid (NTA) column GE Healthcare Discontinued Amicon stirred ultrafiltration cell unit EMD Millipore UFSC40001 CoolSNAP HQ2 Teledyne Photometrics https://www.photometrics.com Ultrasonic cleaner Thermal Fisher Scientific CPX952136R Axopatch 200B amplifier Molecular Devices https://www.moleculardevices.com/ products/axon-patch-clamp-system/ amplifiers

Techniques: Immunoprecipitation, Injection, Staining