Journal: Frontiers in Pharmacology

Article Title: Yangjing capsule improves oligoasthenozoospermia by promoting nitric oxide production through PLCγ1/AKT/eNOS pathway

doi: 10.3389/fphar.2023.1056091

Figure Lengend Snippet: Nitric oxide (NO) concentrations and protein expression of PLCγ1/AKT/eNOS signaling in the testis. (A) NO levels in the testes were measured by using the corresponding commercial kit. (B–E) Protein expression of p-PLCγ1, p-AKT and p-eNOS in the testis. Values are shown as averages ± SD ( n = 6). ## p < 0.01 versus Saline group; * p < 0.05, ** p < 0.01 versus Ornidazole group.

Article Snippet: Then, the samples were incubated with primary antibodies against p-PLCγ1 (Tyr783; Affinity; AF3210; 1:100) or SOX9 (Abcam; ab185966; 5 μg/mL) overnight at 4°C.

Techniques: Expressing, Saline

Journal: Frontiers in Pharmacology

Article Title: Yangjing capsule improves oligoasthenozoospermia by promoting nitric oxide production through PLCγ1/AKT/eNOS pathway

doi: 10.3389/fphar.2023.1056091

Figure Lengend Snippet: Changes of PLCγ1/AKT/eNOS signaling in ornidazole-stimulated primary Sertoli cells. (A–D) Protein levels of p-PLCγ1, p-AKT and p-eNOS were determined using western blot analysis. (E, F) Representative images and quantification of p-PLCγ1 levels in the immunofluorescence assay. Values are shown as averages ± SD ( n = 6). ## p < 0.01 versus PBS group; * p < 0.05, ** p < 0.01 versus Ornidazole + YC (0) group.

Article Snippet: Then, the samples were incubated with primary antibodies against p-PLCγ1 (Tyr783; Affinity; AF3210; 1:100) or SOX9 (Abcam; ab185966; 5 μg/mL) overnight at 4°C.

Techniques: Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Yangjing capsule improves oligoasthenozoospermia by promoting nitric oxide production through PLCγ1/AKT/eNOS pathway

doi: 10.3389/fphar.2023.1056091

Figure Lengend Snippet: PLCγ1-siRNA compromises YC-induced improvement in primary Sertoli cells exposed to ornidazole. (A) PLCγ1-siRNA transfection efficiency was measured using the western blot assay. (B–E) Protein expression of p-PLCγ1, p-eNOS and p-AKT in the western blot analysis. (F, G) Representative images and quantification of p-PLCγ1 levels in the immunofluorescence assay. Values are shown as averages ± SD ( n = 6). ** p < 0.01; ns, not significant.

Article Snippet: Then, the samples were incubated with primary antibodies against p-PLCγ1 (Tyr783; Affinity; AF3210; 1:100) or SOX9 (Abcam; ab185966; 5 μg/mL) overnight at 4°C.

Techniques: Transfection, Western Blot, Expressing, Immunofluorescence

Journal: Journal of cell science

Article Title: Microtubule-associated protein-4 controls nanovesicle dynamics and T cell activation

doi: 10.1242/jcs.199042

Figure Lengend Snippet: (A) mRNA levels of IL2 and CD69 (n=8 and 6) from control and MAP4KD cells activated with SEE-pulsed APCs (4 h). mRNA levels were normalized to a housekeeping gene (ACTB/GAPDH) and to the non-stimulated cells. *P<0.05; **P<0.01 (Wilcoxon test). (B) IL2 secretion levels (pg per ml and 106 cells) measured by ELISA in control and MAP4KD Jurkat T cells activated with SEE-APCs (left panel; n=7) or primaryT lymphoblasts activated with anti-CD3 and -CD28-coated plates (right panel; n=4) for 24 h. *P<0.05; **P<0.01 (paired t-test). (C) CD69 surface expression in control and MAP4KD cells activated with SEE-pulsed APCs (24 h) (n=6). **P<0.01 (paired t-test). (D) Left panel, immunoblot showing PLCγ1 phosphorylation in control and MAP4KD cells conjugated with SEE-APCs. Tubulin is shown as a loading control. Right panel, quantification of PLCγ1 phosphorylation for experiments as shown in the left panel (n=6). *P<0.05; ns, not significant (paired t-test). (E) Immunoblot showing PLCγ1 phosphorylation (PLCγ1 Y873) in control and MAP4KD primary T lymphoblasts activated with crosslinked anti-CD3 and -CD28 antibodies. Tubulin is shown as a loading control, with the quantification under bands (one representative gel out of three). (F) Left panel, images show conjugates of control and MAP4KD Jurkat T cells expressing PKCθ-C1–GFP and activated with SEE-APCs (15 min). Actin (magenta) and bright-field image with CMAC in cyan (APCs) are shown. Scale bar: 10 μm. Right panel, quantification of PKCθ-C1–GFP accumulation for experiments as in the left panel is shown (n=63 and 75 from three independent experiments). **P<0.01 (Mann–Whitney test). (G) Quantification of NFAT (left panel) and NF-κB (right panel) activity as assessed with a luciferase assay (n=6 and 5, respectively). *P<0.05; ***P<0.001 (paired t-test). All graphs represent mean±s.d.

Article Snippet: Antibodies used in this study were: anti-CD3ζ-pY83 (ab68236; 1:1000), anti-CD3ζ (ab190728 1:1000), anti-MAP4 [ab89650;1:800 western blotting (WB), 1:200 immunofluorescence (IF)]; anti-LAT-pY132 (ab4476 1:1000); anti-LAT-pY191 (ab59197 1:1000) (all Abcam); FITC-conjugated anti-α-tubulin (F2168; 1:100), anti-α-tubulin (T6199; 1:2000 WB), anti-β-actin (A2228; 1:1000) (all Sigma); anti-PKCθ (610090; 1:1000), anti-CD4 V450 (560346; 1:100 FACS), anti-CD3ε V500 (561416; 1:100), FITC-conjugated anti-Vβ8 (catalog number: 555606), 1:100), anti-human CD28 (555725, 3 μg/ml) (all BD-Pharmingen); anti-ERK1/2-pT202/Y204 (44285; Calbiochem; 1:1000); anti-p65 (sc372; 1:1000) (all Santa Cruz Biotechnology); anti-PKCθ-pT538 (9377 S; 1:1000), anti-PLCγ1 (2822S; 1:1000), anti-PLCγ1-pY783 (#2821L; 1:1000), anti-ERK1/2 (91075; 1:1000) (all Cell Signaling); anti-SMC1 (A300-055, 1:100) (Bethyl); anti-human CD3ε (300314, 5 μg/ml) and anti-GAPDH (649202,1:1000) (both Biolegend); Percp-Cy5.5-conjugated anti-CD19 (65-0199; 1:100); GHOST Dye Violet 510 (13-0870; 1:500 FACS) (TONBO Biosciences); FITC-conjugated anti-CD69 (21620693; 1:100) (ImmunoTools), and anti-detyrosinated tubulin (Tub Glu; Andrés-Delgado et al., 2012 ).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, MANN-WHITNEY, Activity Assay, Luciferase

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Schematic showing cooling/rewarming sequence in wild-type mice and TrkB signaling on cooling leading to RBM3 and RTN3 expression. (B) Western blot analysis of TrkB signaling shows activation of PLCγ1-CREB pathway in control, cooled and rewarmed mice. TrkB Tyr 516 ( P = 0.0004); TrkB Tyr 816 ( P = 0.0003); CREB Ser 133 ( P < 0.0001); RBM3 ( P = 0.0101); RTN3 ( P = 0.0405). (C) Western blots showing TrkB activation of AKT and ERK branches. Cooling reduced p-ERK and increased p-AKT on cooling. AKT ( P = 0.0278); ERK ( P = 0.0011). (B, C) Representative Western blots of (B) and (C) belong to the same set of experiments with loading controls GAPDH or Actin used for quantification. Bar charts show mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA and Tukey’s multiple comparisons test. For all experiments, n = 4–6 mice per condition.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: Sequencing, Expressing, Western Blot, Activation Assay, Control

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Schematic showing the cold-induced BDNF-TrkB pathway and intervention with the TrkB agonist 7,8-DHF. (B) Western blots showing activation of TrkB signaling in mice after treatment with the TrkB agonist 7,8-DHF (5 mg/kg). Quantification of Western blots (right). All bar charts show mean ± SEM. One-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, N.S., not significant, TrkB Tyr 816 control + vehicle versus 7,8-DHF, 1 h and control + vehicle versus 7,8-DHF, 2 h ( P = 0.0055 and P = 0.0043); RBM3 control + vehicle versus 7,8-DHF, 3 h ( P = 0.0443); RTN3 control + vehicle versus 7,8-DHF, 2 h and control + vehicle versus 7,8-DHF, 3 h ( P = 0.0269 and P = 0.0042); Kruskal–Wallis test and Dunn’s multiple comparisons test, Tyr 783 PLCγ1 control + vehicle versus 7,8-DHF, 1 h and control + vehicle versus 7,8-DHF, 2 h ( P = 0.0066 and P = 0.0341). n = 6 mice per time point.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: Western Blot, Activation Assay, Control

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Schematic showing the in vivo modulation of TrkB signaling on cooling: genetic modulation is via Ntrk2 +/− mice that are hemizygous for TrkB receptor and ACREB; pharmacological antagonism is via ANA-12. (B) TrkB haploinsufficiency abrogated cold-dependent RBM3 induction with a failure to activate the pattern of TrkB signaling seen in control mice with normal TrkB expression. Western blots and bar charts showing markers of the TrkB pathway in Ntrk2 +/+ and Ntrk2 +/− samples pre and post cooling. RBM3 ( P < 0.0001). TrkB Tyr 516; TrkB Tyr 816 are shown as total normalized directly to actin or as relative levels normalized to the basal level of TrkB receptor. (C) TrkB antagonist ANA-12 prevented RBM3 induction on cooling. One-way ANOVA, and Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, N.S., not significant, TrkB Tyr 816 ( P = 0.0008); PLCγ1 Tyr 783 ( P = 0.0007); RBM3 ( P = 0.0074). Bar charts show mean ± SEM. For all experiments, n = 6 mice per time point. Source data are available for this figure.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: In Vivo, Control, Expressing, Western Blot

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Dominant negative inhibitor ACREB prevents CREB phosphorylation and RBM3 induction on cooling. Infection with adeno-associated virus (AAV)-GFP-T2A-ACREB prevented the induction of RBM3 on cooling in vivo. Upstream TrkB pathway is activated independent of CREB inhibition but p-ERK inhibition is prevented. Quantification of Western blots (right). All bar charts show mean ± SEM. One-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, N.S., not significant, TrkB Tyr 816 (AAV-empty control versus AAV-empty cooled [ P = 0.0028]; AAV-empty control versus AAV-ACREB cool [ P = 0.0023]); Tyr 783 PLCγ1 (AAV-empty control versus AAV-empty cooled [ P = 0.0035]); Thr202/Thr204 ERK (AAV-empty control versus AAV-empty cooled [ P = 0.0343]); S133 CREB (AAV-empty control versus AAV-empty cooled [ P = 0.0382], AAV-empty cooled versus AAV-ACREB cooled [ P = 0.0005]); RBM3 (AAV-empty control versus AAV-empty cooled [ P = 0.0073]). n = 6 mice per condition. (B) RBM3 controls the induction of the ERK-specific phosphatase Dusp6 on cooling in vitro in Rbm3 −/0 neurons. Western blot shows Dusp6 increase on cooling of wild-type neurons. Rbm3 −/0 neurons showed no increase in Dusp6 on cooling. Bar chart shows quantification of Western blots (right); mean ± SEM. Two-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, N.S., not significant, Dusp6 wild-type control versus wild-type cooled ( P = 0.0299); Rbm3 −/0 cooled versus Rbm3 −/0 rewarmed ( P = 0.0028). n = 5 cultures per condition. (C) ERK inhibitors U0126 and PD98059 reduce ERK phosphorylation on cooling in vitro in Rbm3 −/0 neurons. Representative Western blots showing the effect of U0126 and PD98059 inhibitors on ERK Thr202/Thr204 phosphorylation. Wild-type and Rbm3 −/0 neurons at 37°C (white bar), or 32°C (blue bar) untreated, or treated with vehicle or the respective ERK inhibitors U0126 and PD98059. (D) ANA-12 antagonist prevents TrkB-mediated RBM3 induction in prion diseased mice. Representative Western blots showing inhibition of TrkB phosphorylation (Y816) and induction of RBM3 mediated by ANA-12 TRkB antagonist. Bar charts quantifications. Two-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, N.S., not significant, TrkB Tyr 816 control + vehicle versus vehicle cooled + vehicle ( P = 0.0003), cooled + vehicle versus cooled + ANA-12 ( P = 0.0025); RBM3 (control + vehicle versus cooled + vehicle [ P = 0.0020], cooled + vehicle versus cooled + ANA-12 [ P = 0.0137]). n = 6 mice per condition. Source data are available for this figure.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: Dominant Negative Mutation, Phospho-proteomics, Infection, Virus, In Vivo, Inhibition, Western Blot, Control, In Vitro

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Schematic shows primary hippocampal neurons from Rbm3 −/0 and wild-type mice cultured for 21 d and subjected to cooling at 32°C for 24 h before rewarming. Western blots show TrkB signaling on cooling in wild-type neurons as before. Rbm3 −/0 neurons showed high levels of p-ERK, not low, and absent RBM3 expression. Graphs show quantification of Western blots (right). Two-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05 (TrkB Tyr 816 wild-type control versus cool P = 0.0289; Tyr 783 PLCγ1 P = 0.0444; Thr202/Thr204 ERK wild-type cooled versus RBM3 −/0 P = 0.0137; RBM3 wild-type control versus wild-type cooled P = 0.0020) and Dunnett’s multiple comparisons test (S133 CREB wild-type control versus wild-type cooled P = 0.0234), N.S., not significant. n = 3–5 cultures per condition. (B) Primary hippocampal neurons cultured from Rbm3 −/0 mice show impaired synaptic plasticity on cooling and rewarming. Representative confocal images of control, cooled, and rewarmed neurons are shown. Neurons were immunostained with the pre-synaptic marker, synaptophysin (magenta), and the post-synaptic marker, PSD-95 (green). White arrowheads point toward synapses. Scale bar, 5 μm. (top). Bar graph shows mean normalized synapse number ± SEM. One-way ANOVA (bottom left) and Tukey’s multiple comparisons test, * P < 0.05, *** P < 0.001; N.S., not significant; (wild-type control versus wild-type cooled, P ≤ 0.0001, wild-type cooled versus wild-type rewarmed, P ≤ 0.0001; wild-type rewarmed versus Rbm3 −/0 rewarmed P = 0.0132). n = 3 cultures per condition. (C) Representative electron microscopy micrographs of wild-type and Rbm3 −/0 mice that have been cooled then rewarmed. The presynaptic compartment is pseudo-colored yellow and post-synaptic compartment green. Lack of RBM3 expression abrogates synapse recovery observed in wild-type mice in the CA1 region of the hippocampus. Red stars indicate synapses with pseudocoloured pre- (yellow) and post- (green) synaptic compartments. Bar chart showing mean ± SEM quantification from three animals (93 images) per condition. Two-tailed t test ** P < 0.01 ( P = 0.0051). Scale bar, 1 μm. n = 3 mice per condition. (D) ERK inhibitors restored capacity for synapse dismantling in Rbm3 −/0 neurons. Representative confocal images are shown. Neurons were cooled at 32°C (24 h) and treated with vehicle (control), U0126 (10 μM, 5 h), or PD98059 (25 μM, 5 h). Neurons were immunostained with the pre-synaptic marker, synaptophysin (magenta), and the post-synaptic marker, PSD-95 (green). White arrowheads point toward synapses. Scale bar, 5 μm. Bar graph shows mean normalized synapse number ± SEM. * P < 0.05, *** P < 0.001; N.S., not significant; one-way ANOVA (right); ( P = 0.0221 and P < 0.0001 and Tukey’s multiple comparisons test). n = 3 cultures per condition.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: Cell Culture, Western Blot, Expressing, Control, Marker, Electron Microscopy, Two Tailed Test

Journal: Life Science Alliance

Article Title: TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection

doi: 10.26508/lsa.202000884

Figure Lengend Snippet: (A) Primary hippocampal neurons were cultured for 19–20 d and subjected to cooling at 32°C for 24 h before re-warming. Blocking TrkB activation using the ligand scavenger TrkB-Fc prevented TrkB-PLCγ1-CREB signaling and RBM3 induction on cooling. Control IgG treated neurons showed pathway activation as wild-type mice . Total levels of synaptic protein PSD95 are unchanged. Bar charts show mean ± SEM. Two-way ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, TrkB Tyr 816 ( P = 0.0014); Tyr 516 ( P = 0.0025); Tyr 783 PLCγ1 ( P = 0.0043); CREB Ser 133 ( P = 0.0218); RBM3 ( P = 0.0036). n = 3–5 cultures per condition. (B) TrkB-Fc prevented synapse recovery following cooling. Confocal images of representative dendritic fragments labeled for the presynaptic protein synaptophysin (magenta) and the post-synaptic protein PSD95 (green). White arrowheads point toward synapses. Top panels show control IgG-treated, bottom panels show TrkB-Fc treated samples. Bar chart shows quantification of co-localized synaptophysin and PSD95 puncta (right). One-way ANOVA, * P < 0.05, ** P < 0.01, (C-IgG control versus C-IgG cooled < 0.0001; C-IgG cooled versus C-IgG rewarmed P = 0.0014; C-IgG rewarmed versus TrkB-Fc rewarmed P = 0.0027). Bar chart shows numbers of synaptophysin particles. One-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, (C-IgG cooled versus TrkB-Fc cooled P = 0.0182; C-IgG cooled versus TrkB-Fc rewarmed P = 0.0044; C-IgG rewarmed versus TrkB-Fc cooled P = 0.0109; C-IgG rewarmed versus TrkB-Fc rewarmed P = 0.0025; TrkB-Fc control versus TrkB-Fc cooled P < 0.0001). n = 3 cultures per condition. Scale bar, 10 μm. (C) The TrkB-specific antagonist ANA-12 impairs synapse recovery on rewarming after cooling in wild-type mice. Representative EM micrographs and graph bar are shown. Red stars indicate synapses; which have been pseudo-colored pre- (yellow) and post- (green) synaptic compartments. * P < 0.05, t test (vehicle versus ANA-12 P = 0.0466). n = 3 mice per condition.

Article Snippet: Proteins were detected using the following antibodies: TrkB, p-TrkB γ816, and p-TrkB γ516 (1:1,000; catalog number: 4603, 4168, 4619; Cell Signaling Technology); PLCγ1 and p-PLCγ1 γ783 (1:1,000; catalog number: 2822 and 2821; Cell Signaling Technology); AKT and p-AKT Ser473 (1:1,000; catalog number: 9272, 9271; Cell Signaling Technology), p44/42 MAPK (Erk1/2) and p-p44/42 MAPK (p-Erk1/2) (1:1,000; catalog number: 4695, 4376; Cell Signaling Technology); CREB and p-CREB Ser133 (1:1,000; catalog number: 9104, 9198; Cell Signaling Technology); RBM3 (1:1,000; catalog number: 14363-1-AP; ProteinTech); RTN3 (custom made in rabbit against RTN3 peptide 213–227 a.a. H-CARDQTKSIVEKIQAK-NH2 [Sigma-Aldrich] by UKSBS); MKP3/Dusp6 (1:1,000; catalog number: sc-377070; Santa Cruz Technology), and PSD95 (1:1,000; catalog number: 04-1066; Millipore).

Techniques: Cell Culture, Blocking Assay, Activation Assay, Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Effects of E7050 on VEGFR2-mediated signaling pathways in VEGF-stimulated HUVECs. Cells were serum-starved for 6 h and then pretreated with E7050 (5 and 10 μM) or vehicle for 1 h, followed by stimulation with VEGF (100 ng/mL) for another 10 min (VEGFR2, PLCγ1, FAK, and Src) or 30 min (Akt, JNK, and p38 MAPK) before proteins were collected. The expression and phosphorylation status of VEGFR2 and its downstream effectors, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK, were detected by Western blotting using respective antibodies. ( A ) E7050 inhibited the phosphorylation (Tyr1175) of VEGFR2 induced by VEGF in HUVECs. ( B ) The quantified results show that the ratio of p-VEGFR2 protein normalized to the total amount of VEGFR2 protein, which was measured by densitometry. ( C ) E7050 inhibited the phosphorylation of PLCγ1, FAK, and Src in VEGF-stimulated HUVECs. Calculated ratios of ( D ) p-PLCγ1, ( E ) p-FAK, and ( F ) p-Src normalized to the relative total protein levels are shown. ( G ) E7050 inhibited the phosphorylation of Akt, JNK, and p38 MAPK in VEGF-stimulated HUVECs. The compiled results of the ratios of ( H ) p-Akt, ( I ) p-JNK, and ( J ) p-p38 MAPK normalized to the relative total protein levels are shown. The data are presented as mean ± SEM of three independent experiments. # p < 0.05 compared with the untreated cells. * p < 0.05 compared with the vehicle-treated cells.

Article Snippet: Primary antibodies against VEGFR2 (#2479; 1:20,000 dilution), phospho (p)-VEGFR2 (Tyr1175) (#2478; 1:1500 dilution), Akt (#4691; 1:20,000 dilution), p-Akt (Ser473) (#4060; 1:5000 dilution), FAK (#3285; 1:7000 dilution), p-FAK (Tyr397) (#8586; 1:1500 dilution), PLCγ1 (#5690; 1:8000 dilution), p-PLCγ1 (Ser1248) (#8713; 1:2500 dilution), Src (#2109; 1:30,000 dilution), p-Src (Tyr416) (#6943; 1:6000 dilution), JNK (#9258; 1:1000 dilution), p-JNK (Thr183/Tyr185) (#4668; 1:1000 dilution), ERK1/2 (#4695; 1:15,000 dilution), p-ERK1/2 (Thr202/Tyr204) (#4370; 1:15,000 dilution), p38 MAPK (#9212; 1:12,000 dilution), and p-p38 MAPK (Thr180/Tyr182) (#4511; 1:2000 dilution) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: E7050 Suppresses the Growth of Multidrug-Resistant Human Uterine Sarcoma by Inhibiting Angiogenesis via Targeting of VEGFR2-Mediated Signaling Pathways

doi: 10.3390/ijms24119606

Figure Lengend Snippet: Schematic diagram of a proposed mechanism of E7050-induced anti-angiogenic activity. E7050 exerts anti-angiogenic effects in VEGF-stimulated endothelial cells by downregulating the phosphorylation of VEGFR2 and its downstream mediators, including PLCγ1, FAK, Src, Akt, JNK, and p38 MAPK. The blockage of VEGFR2-mediated signaling cascade pathways by E7050 contributes to the inhibition of proliferation, migration, and tube formation in endothelial cells.

Article Snippet: Primary antibodies against VEGFR2 (#2479; 1:20,000 dilution), phospho (p)-VEGFR2 (Tyr1175) (#2478; 1:1500 dilution), Akt (#4691; 1:20,000 dilution), p-Akt (Ser473) (#4060; 1:5000 dilution), FAK (#3285; 1:7000 dilution), p-FAK (Tyr397) (#8586; 1:1500 dilution), PLCγ1 (#5690; 1:8000 dilution), p-PLCγ1 (Ser1248) (#8713; 1:2500 dilution), Src (#2109; 1:30,000 dilution), p-Src (Tyr416) (#6943; 1:6000 dilution), JNK (#9258; 1:1000 dilution), p-JNK (Thr183/Tyr185) (#4668; 1:1000 dilution), ERK1/2 (#4695; 1:15,000 dilution), p-ERK1/2 (Thr202/Tyr204) (#4370; 1:15,000 dilution), p38 MAPK (#9212; 1:12,000 dilution), and p-p38 MAPK (Thr180/Tyr182) (#4511; 1:2000 dilution) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Inhibition, Migration

Journal: Frontiers in Aging Neuroscience

Article Title: Exploring the neuroprotective mechanism of Tongqiao Huashuan granules in vascular dementia based on PLCγ1/IP3R signaling pathway

doi: 10.3389/fnagi.2026.1719664

Figure Lengend Snippet: TQHS increased PLCγ1 expression, whereas reduced CaM expression in the hippocampal CA1 and CA3 regions of VaD Rats. (A) Immunohistochemical analysis of PLCγ1 and CaM expression in hippocampal CA1 and CA3 regions (× 10) ( n = 2). (B–E) Quantitative analysis showed the expression of (B) PLCγ1, and (D) CaM in hippocampal CA1 regions and (C) PLCγ1, and (E) CaM in hippocampal CA3 regions. Values were expressed as means ± SD ( n = 3). ## P < 0.01, ### P < 0.001 versus sham-surgery group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus model group.

Article Snippet: Following washes and blocking (5% skim milk, 1 h), membranes were probed overnight (4°C) with primary antibodies against p-PLCγ1 (1:2,000 dilution, AF3210, AB_2834502, Affinity Biosciences, United States), PLCγ1 (1:2,000 dilution, AF6210, AB_2835091, Affinity Biosciences, United States), IP3R (1:2000 dilution, DF3000, AB_2840979, Affinity Biosciences, United States), CAMKKII (1:1,000 dilution, DF4793, AB_2837147, Affinity Biosciences, United States), GAPDH (1:50,000 dilution, 60004-1-Ig, AB_2107436, Proteintech, China), CaM (1:2,000 dilution, AF6353, AB_2835158, Affinity Biosciences, United States) and β-actin (1:10,000 dilution, AF7018, AB_2839420, Affinity Biosciences, United States).

Techniques: Expressing, Immunohistochemical staining

Journal: Frontiers in Aging Neuroscience

Article Title: Exploring the neuroprotective mechanism of Tongqiao Huashuan granules in vascular dementia based on PLCγ1/IP3R signaling pathway

doi: 10.3389/fnagi.2026.1719664

Figure Lengend Snippet: TQHS increased PLCγ1, p-PLCγ1, IP3R and CAMKKII expression, whereas reduced CaM expression in the hippocampus of VaD rats. (A,F) Western blot was performed to detect the levels of PLCγ1, p-PLCγ1, IP3R, CAMKKII and CaM in the hippocampus of VaD rats. Quantitative analysis of (B) PLCγ1, (C) CAMKKII, (D) p-PLCγ1, (E) IP3R and (G) CaM expression. The densities of the bands were normalized with respect to the values of GADPH and β-actin. Values were expressed as means ± SD ( n = 3). ### P < 0.001 versus sham-surgery group. ** P < 0.01, *** P < 0.001 versus model group.

Article Snippet: Following washes and blocking (5% skim milk, 1 h), membranes were probed overnight (4°C) with primary antibodies against p-PLCγ1 (1:2,000 dilution, AF3210, AB_2834502, Affinity Biosciences, United States), PLCγ1 (1:2,000 dilution, AF6210, AB_2835091, Affinity Biosciences, United States), IP3R (1:2000 dilution, DF3000, AB_2840979, Affinity Biosciences, United States), CAMKKII (1:1,000 dilution, DF4793, AB_2837147, Affinity Biosciences, United States), GAPDH (1:50,000 dilution, 60004-1-Ig, AB_2107436, Proteintech, China), CaM (1:2,000 dilution, AF6353, AB_2835158, Affinity Biosciences, United States) and β-actin (1:10,000 dilution, AF7018, AB_2839420, Affinity Biosciences, United States).

Techniques: Expressing, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Exploring the neuroprotective mechanism of Tongqiao Huashuan granules in vascular dementia based on PLCγ1/IP3R signaling pathway

doi: 10.3389/fnagi.2026.1719664

Figure Lengend Snippet: Effects of TQHS granules on OGD/R-induced cytotoxicity SH-SY5Y cells and the mRNA expressions of PLCγ1, IP3R, and TrkB. (A) Effects of U73122 on normal SH-SY5Y cell viability. (B,C) Effects of B-U73122 and (C) TQHS granules on OGD/G cell viability. (D–F) RT-qPCR analyzed (D) TrkB, (E) IP3R and (F) PLCγ1 mRNA expression levels. Values were expressed as means ± SD ( n = 3). ## P < 0.01, ### P < 0.001 versus control group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus OGD/R group.

Article Snippet: Following washes and blocking (5% skim milk, 1 h), membranes were probed overnight (4°C) with primary antibodies against p-PLCγ1 (1:2,000 dilution, AF3210, AB_2834502, Affinity Biosciences, United States), PLCγ1 (1:2,000 dilution, AF6210, AB_2835091, Affinity Biosciences, United States), IP3R (1:2000 dilution, DF3000, AB_2840979, Affinity Biosciences, United States), CAMKKII (1:1,000 dilution, DF4793, AB_2837147, Affinity Biosciences, United States), GAPDH (1:50,000 dilution, 60004-1-Ig, AB_2107436, Proteintech, China), CaM (1:2,000 dilution, AF6353, AB_2835158, Affinity Biosciences, United States) and β-actin (1:10,000 dilution, AF7018, AB_2839420, Affinity Biosciences, United States).

Techniques: Quantitative RT-PCR, Expressing, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Exploring the neuroprotective mechanism of Tongqiao Huashuan granules in vascular dementia based on PLCγ1/IP3R signaling pathway

doi: 10.3389/fnagi.2026.1719664

Figure Lengend Snippet: Potential mechanism underlying the neuroprotective effects of TQHS granules in VaD rats and SH-SY5Y cells. TQHS granules improved cognitive function in VaD rats, generated IP3 through PLCγ1-mediated catalytic hydrolysis of PIP2, and then affected the expression of IP3R downstream of the pathway, thus protecting the brain from VaD-induced oxidative stress and calcium overload, and further induced antioxidant activity and regulated intracellular calcium overload mechanisms.

Article Snippet: Following washes and blocking (5% skim milk, 1 h), membranes were probed overnight (4°C) with primary antibodies against p-PLCγ1 (1:2,000 dilution, AF3210, AB_2834502, Affinity Biosciences, United States), PLCγ1 (1:2,000 dilution, AF6210, AB_2835091, Affinity Biosciences, United States), IP3R (1:2000 dilution, DF3000, AB_2840979, Affinity Biosciences, United States), CAMKKII (1:1,000 dilution, DF4793, AB_2837147, Affinity Biosciences, United States), GAPDH (1:50,000 dilution, 60004-1-Ig, AB_2107436, Proteintech, China), CaM (1:2,000 dilution, AF6353, AB_2835158, Affinity Biosciences, United States) and β-actin (1:10,000 dilution, AF7018, AB_2839420, Affinity Biosciences, United States).

Techniques: Generated, Expressing, Antioxidant Activity Assay