pkcγ dn  (Addgene inc)

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: Figure 2. TRPM2-mediated Ca2+ influx promotes PKCg activation (A) FRET sensor CKAR consists of a CFP, a YFP, and a linker. Without PKC activity, CFP and YFP are in close proximity (<10 nm) for FRET (emission of CFP by 440 nm excitation excites YFP and yields a 527 emission). PKC phosphorylates CKAR, which changes the conformation of the linker and eliminates FRET (490 nm emission of CFP cannot excite YFP). (B–D) CKAR real-time imaging in HEK293T cells transfected with TRPM2/PKCg and EGFP/PKCg. (B) FRET intensity view before and 10 min after 100 mM H2O2 perfusion. (C) Averaged representative traces from 5 randomly chosen cells. (D) Quantification of FRET changes (n = 10–20). (E) Graphic illustration showing where EGTA, ACA, and BAPTA-AM target. (F–H) CKAR real-time imaging in HEK293T cells transfected with TRPM2/PKCg pretreated with ACA (10 mM) or BAPTA-AM (10 mM) for 30 min and perfused with Ca2+ free extracellular solution buffered by 2 mM EGTA. (F) FRET intensity before and 10 min after 100 mM H2O2 perfusion. (G) Averaged representative traces from 5 randomly chosen cells. (H) Quantification of FRET changes (n = 10–20). ***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: Activation Assay, Activity Assay, Imaging, Transfection

Journal: Cell reports

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

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: Figure 3. PKCg binding motif in TRPM2 is required for TRPM2-PKCg coupling (A) Alignment of the PKC binding sequence in RACK and annexin1 with human TRPM2. (B) Co-immunoprecipitation of PKCg by TRPM2 in HEK293T cells co-expressed with PKCg and TRPM2, N tail of TRPM2 (TRPM2-NT), and PKC-binding-motif- deleted TRPM2 (TRPM2-DPBM). (C) Graphic illustration showing the structure of TRPM2 and the position of PBM. (D) In vitro binding assay. The C2 domain of PKCg was conjugated with a GST tag, while the MHR1/2 domain of TRPM2 was conjugated with a His6 tag. Anti-GST antibody was used for immunoprecipitation, and GST was used as a negative control (labeled as #2). Pull-down efficiency was evaluated using Coomassie blue staining (left) and immunoblotting by anti-His6 antibody (right). (E and F) Whole-cell current recording of TRPM2 in HEK293T cells transfected with PKCg and TRPM2-WT (green) or TRPM2-DPBM (red). (E) Representative traces. PMA (10 mM) was used to induce TRPM2 activation, NMDG to test seal tightness, and ACA to block TRPM2 current. (F) Quantification of current amplitude (n = 9, 8). (G–I) CKAR real-time imaging in HEK293T cells transfected with PKCg and TRPM2-WT (green) or TRPM2-DPBM (red). (G) FRET intensity before and 10 min after 100 mM H2O2 perfusion. (H) Averaged representative traces from 5 randomly chosen cells. (I) Quantification of FRET changes (n = 10–20).

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: Binding Assay, Sequencing, Immunoprecipitation, In Vitro, Negative Control, Labeling, Staining, Western Blot, Transfection, Activation Assay, Blocking Assay, Imaging