Journal: Cell reports

Article Title: TRPM2 enhances ischemic excitotoxicity by associating with PKCγ

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: (A) Graphic illustration showing how TAT-M2PBM works. The PKCγ binding motif of TRPM2 (M2PBM) in conjugation with the cell-penetrating peptide TAT was synthesized. TAT-M2PBM binds to the TRPM2 binding site for PKCγ, thus achieving the competitive inhibition on the binding of TRPM2 to PKCγ. (B) Co-immunoprecipitation of PKCγ by TRPM2 in HEK293T cells co-expressed with PKCγ and TRPM2 treated with TAT-SC (scramble) or TAT-M2PBM at 1 μM for 2 h. (C and D) Whole-cell current recording of TRPM2 in HEK293T cells transfected with PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). (C) Representative traces. PMA (10 μM) was used to induce TRPM2 activation, NMDG to test seal tightness, and ACA to block TRPM2 current. (D) Quantification of current amplitude (n = 9, 9). (E and F) CKAR real-time imaging in HEK293T cells transfected with PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). (E) Averaged representative traces from 5 randomly chosen cells. (F) Quantification of FRET changes (n = 10–20). (G and H) Whole-cell current recording of NMDARs in HEK293T cells transfected with NMDARs/PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). (G) Representative traces. NMDA (100 μM) was used to induce NMDAR activation. (H) Quantification of current amplitude (n = 11, 10, 11). (I and J) Whole-cell current recording of NMDARs in neurons isolated from the WT and M2KO mice treated with TAT-SC or TAT-M2PBM. (I) Representative traces. (J) Quantification of current amplitude (n = 15, 11, 9, 6). (K and L) Surface expression of GluN2a and GluN2b in HEK293T cells transfected with NMDAR/PKCγ and TRPM2 treated with TAT-SC or TAT-M2PBM. (K) Representative western blot (WB) bands. (L) Quantification of relative expression normalized to pan-cadherin (n = 6/group). (M–O) Whole-cell current recording of NMDARs in neurons isolated from the WT and M2KO mice treated with TAT-SC or TAT-M2PBM. (M) Representative traces. PMA at 10 μM was used to enhance NMDAR’s activity for 60 s. (N) Quantification of current amplitude before and after PMA perfusion. (O) Quantification of current increases after PMA perfusion (n = 6–10/group). ns, no statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001; unpaired t test; mean ± SEM.

Article Snippet: Rabbit polyclonal antibodies to TRPM2 (Novus, NB110–81601, 1:50 in protein extraction for IP); Rabbit polyclonal antibodies to GluN2A (Cell Signaling Technology, 4205S, 1:1000 in 5% BSA for WB).

Techniques: Binding Assay, Conjugation Assay, Synthesized, Inhibition, Immunoprecipitation, Transfection, Activation Assay, Blocking Assay, Imaging, Isolation, Expressing, Western Blot, Activity Assay

Journal: Cell reports

Article Title: TRPM2 enhances ischemic excitotoxicity by associating with PKCγ

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: (A) Co-immunoprecipitation of PKCγ, 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-EE 3 , 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: Rabbit polyclonal antibodies to TRPM2 (Novus, NB110–81601, 1:50 in protein extraction for IP); Rabbit polyclonal antibodies to GluN2A (Cell Signaling Technology, 4205S, 1:1000 in 5% BSA for WB).

Techniques: Immunoprecipitation, Injection, Staining

Journal: Cell reports

Article Title: TRPM2 enhances ischemic excitotoxicity by associating with PKCγ

doi: 10.1016/j.celrep.2024.113722

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal antibodies to TRPM2 (Novus, NB110–81601, 1:50 in protein extraction for IP); Rabbit polyclonal antibodies to GluN2A (Cell Signaling Technology, 4205S, 1:1000 in 5% BSA for WB).

Techniques: Virus, Recombinant, Sequencing, Bicinchoninic Acid Protein Assay, Isolation, Membrane, Extraction, Protein Extraction, Knock-Out, Mutagenesis, Subcloning, Plasmid Preparation, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: Rescue of sharp wave-ripples and prevention of network hyperexcitability in the ventral but not the dorsal hippocampus of a rat model of fragile X syndrome

doi: 10.3389/fncel.2023.1296235

Figure Lengend Snippet: Western blotting analysis of the three NMDAR subunits, NR1, NR2A, and NR2B in WT and KO dorsal (A) and ventral hippocampus (B) . The results of the statistical analysis (independent t -test) showed similar expressions between the WT and KO dorsal hippocampus (NR1: t 14 = 0.107, p = 0.813; NR2A: t 14 = –0.24, p = 0.369; NR2B: t 14 = 1.128, p = 0.842; n = 8 rats in either genotype), and ventral hippocampus (NR1: t 14 = –0.241, p = 0.813; NR2A: t 14 = 0.803, p = 0.435; NR2B: t 14 = 0.203, p = 0.842; n = 8 rats in either genotype). Also, the NR2A/NR2B ratio is similar in WT and KO dorsal ( t 10.15 = –1.38, p = 0.199) and ventral hippocampus ( t 9.72 = 0.383, p = 0.71). The expression of NR2A subunit and the NR2A/NR2B ratio is higher in the dorsal compared with the ventral hippocampus in both WT (NR2A: t 14 = 4.27, p < 0.001, and NR2A/NR2B ratio: t 14 = 3.08, p = 0.008) and KO rats (NR2A: t 14 = 4.017, p = 0.001, and NR2A/NR2B ratio: t 14 = 3.01, p = 0.009). In contrast, NR1 and NR2B are similarly expressed in WT and KO dorsal (NR1: t 14 = 1.308, p = 0.212, and NR2B: t 14 = 1.09, p = 0.294) and ventral hippocampus (NR1: t 14 = 1.039, p = 0.316, and NR2B: t 14 = 0.187, p = 0.854). (C) Images of individual western blot samples with detected bands of the NMDA receptor protein subunits, and the corresponding loading marker band of beta actin. “ns” denotes not significant difference.

Article Snippet: Membranes were next incubated overnight at 4°C with the following primary antibodies diluted in 3% PBST: rabbit anti-α1 GABA A R polyclonal antibody (1:2500 #06-868, Millipore Sigma), rabbit anti-NR1 monoclonal antibody (1:1000 #D65B7, Cell Signaling), rabbit anti-NR2A polyclonal antibody (1:1000 #4205, Cell Signaling), rabbit anti-NR2B monoclonal antibody (1:1000 #B8E10, Cell Signaling) and rabbit anti-β-actin polyclonal antibody (1:15000 #E-AB-20058, Elabscience).

Techniques: Western Blot, Expressing, Marker

Journal: eLife

Article Title: KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization

doi: 10.7554/eLife.72483

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-GluN2A, (rabbit polyclonal) , Cell Signaling Technology , Cat# 4205, RRID: AB_2112295 , WB (1:2000).

Techniques: Virus, SYBR Green Assay, Staining, Bicinchoninic Acid Protein Assay, Software, Sequencing, Recombinant

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: FKN/NR Signaling Pathway Regulates Hippocampal Inflammatory Responses: the Survival of Hippocampal Neurons in Diabetic Rats with Chronic Unpredictable Mild Stress

doi: 10.1155/2022/8980627

Figure Lengend Snippet: Expression of CX3CR1, FKN, NR2A, and NR2B in neuronal cells under HCS. (a) Expression picture in each group. (b) Comparison of protein expression of CX3CR1, FKN, NR2A, and NR2B in hippocampal neurons of each group under coculture. Note that, compared with the normal group, # P < 0.05 and ## P < 0.01; compared with the model group, ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Rabbit anti-polyclonal CX3CR1, FKN, was purchased from Abcam, UK; rabbit anti-polyclonal NR2A and NR2B antibodies were purchased from CST, USA, while GAPDH was purchased from Proteintech, USA.

Techniques: Expressing, Comparison