Journal: Frontiers in Pharmacology

Article Title: Kaixin-San improves Aβ-induced synaptic plasticity inhibition by affecting the expression of regulation proteins associated with postsynaptic AMPAR expression

doi: 10.3389/fphar.2023.1079400

Figure Lengend Snippet: Effect of KXS on LTP and AMPAR expression. (A) fEPSP waveforms in each group at different stages. (B) Alterations in the fEPSP PS amplitude in each group at different time points. At 80 min post HFS, the PS amplitude in the Aβ/KXS group significantly increased compared with that in the Aβ group. Each value represents the mean ± SE. (C) Statistical differences of mice in three groups at different time points. (D–F) Statistical analysis of the GluR1, GluR2, and GluR2/3 expression performed following Western blotting. The pictures below the graph indicate the protein exposure; n = 6 per group. Each column with a bar represents the mean ± SD. * p < 0.05 versus the control group; # p < 0.05 versus the Aβ group.

Article Snippet: The proteins were transferred onto PVDF membranes via wet transfer using 100 V constant pressure for 2 h. After 1 h of incubation with the Western blocking solution at room temperature, the membranes were incubated with one of the primary antibodies (anti-GluR1 [1:2,000, ab109450; Abcam]; anti-GluR2 [1:2,000, ab206293; Abcam]; anti-phospho-GluR1 [Ser845, ab222761; Abcam]; anti-phospho-GluR2 [Ser880, bs-5359R; Bioss]; anti-GluR2/3 [1:500, AF5458; Affinity]; anti-NSF [1:500, DF4611; Affinity]; anti-GRIP1 [1:500, DF2500; Affinity]; anti-ABP [1:500, bs-2410R; Bioss]; anti-PKA alpha + beta [1:500, bs-0520R; Bioss]; anti-beta-actin [1:5,000, bs-0061R; Bioss]; and anti-GAPDH [1:3,000, bs-2188R; Bioss]) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Kaixin-San improves Aβ-induced synaptic plasticity inhibition by affecting the expression of regulation proteins associated with postsynaptic AMPAR expression

doi: 10.3389/fphar.2023.1079400

Figure Lengend Snippet: Effect of KXS on the levels of AMPAR subunits related to the protein expression. The statistical analysis of ABP (A) , GRIP1 (B) , NSF (C) , pGluR1–Ser845 (D) , pGluR1–Ser845/GluR1 (E) , PKA (α+β) (F) , pGluR2–Ser880 (G) , pGluR2–Ser880/GluR2 (H) , and PKC δ (I) expression was determined using Western blotting. The pictures below the graph indicate the protein exposure; n = 6 per group. Each column with a bar represents the mean ± SD. * p < 0.05 versus the control group; # p < 0.05 versus the Aβ group. During the experiment, GluR1 and pGluR1–Ser845, as well as GluR2 and pGluR2–Ser880, were run simultaneously on the same electrophoretic gel; hence, the same loading control is used in and and in and . The molecular weights of PKA (α + β) and PKC δ are 40 kD and 77 kD, respectively; therefore, they may be separated on one gel and transferred to the same PVDF membrane. The same loading control is used in .

Article Snippet: The proteins were transferred onto PVDF membranes via wet transfer using 100 V constant pressure for 2 h. After 1 h of incubation with the Western blocking solution at room temperature, the membranes were incubated with one of the primary antibodies (anti-GluR1 [1:2,000, ab109450; Abcam]; anti-GluR2 [1:2,000, ab206293; Abcam]; anti-phospho-GluR1 [Ser845, ab222761; Abcam]; anti-phospho-GluR2 [Ser880, bs-5359R; Bioss]; anti-GluR2/3 [1:500, AF5458; Affinity]; anti-NSF [1:500, DF4611; Affinity]; anti-GRIP1 [1:500, DF2500; Affinity]; anti-ABP [1:500, bs-2410R; Bioss]; anti-PKA alpha + beta [1:500, bs-0520R; Bioss]; anti-beta-actin [1:5,000, bs-0061R; Bioss]; and anti-GAPDH [1:3,000, bs-2188R; Bioss]) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Membrane

Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, Extraction, MANN-WHITNEY, Immunostaining

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Expression of KIF5A, GluR2 and beta 2+3 subunits of gamma aminobutyric acid receptors (Gabrb2+3) in an in vivo model of seizures. (A) EEG results: There was no epileptic discharge in the Ctl group after the injection of saline; however, epileptic discharge was observed in the Sez group after the injection of PTZ. (B) Total protein expression: the gray level of the total protein expression bands was normalized with GAPDH, and the total protein expression levels of KIF5A, GluR2 and Gabrb2+3 in the hippocampus of the Sez group did not change significantly (n=6 in each group, vs. Ctl, P>0.05). (C) Surface protein expression: The gray level of the total protein expression bands was normalized with Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1). In the Sez group, the expression of GluR2 on the surface increased significantly to 181.74%±14.44% ( vs. Ctl, # , P<0.01). Conversely, the expression of GluR2 on the surface decreased to 19.62%±8.01% ( vs. Ctl, # , P<0.01). KIF5A, kinesin superfamily proteins 5A; GluR2, glutamate receptors subunit-2; Ctl, control; Sez, seizures; PTZ, pentylenetetrazol.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Expressing, In Vivo, Injection, Saline, Control

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Receptor recycling assay (IF). The recycling ratio of GluR2 was 0.30±0.05 in the Ctl group and 0.60±0.07 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01), and the recycling ratio of Gabrb2+3 was 0.49±0.04 in Ctl group and 0.32±0.05 in the Mg 2+ -free group (7 cells per group, vs. Ctl, # , P<0.01) (×600). Scale Bar: 50 µm. IF, immunofluorescence; Ctl, control.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Immunofluorescence, Control

Journal: Annals of Translational Medicine

Article Title: The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A

doi: 10.21037/atm-22-4337

Figure Lengend Snippet: Interaction between KIF5A and GluR2 and Gabrb2+3 in the seizure model. (A) Co-ip results: The protein bands showed the co-ip levels of KIF5A, GluR2, and Gabrb2+3, and normalized with the protein levels of KIF5A. The GluR2 level of KIF5A pull-down in the hippocampi of the rats in the Sez group increased to 130.42%±53.24% (n=6 per, vs. Ctl, *, P<0.05). However, the Gabrb2+3 level of KIF5A decreased to 50.86%±5.33% in the Sez group (n=6 per group, vs. Ctl, # , P<0.01). (B) IF results: the Pearson’s correlation coefficients (PCC) of KIF5A/GluR2 was 0.40±0.19 in the Ctl group and 0.87±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. (C) IF results: the PCC of KIF5A/Gabrb2+3 was 0.97±0.02 in the Ctl group and 0.32±0.11 in the Mg 2+ -free solution group (n=6 per group, vs. Ctl, # , P<0.01) (×400). Scale Bar: 100 µm. IF, immunofluorescence.

Article Snippet: The primary antibodies used include anti-KIF5A antibody 1:1,000 (SANTA, sc-376452), anti-GluR2 antibody 1:1,000 (BOSTER, PB9205), and anti-GABAaR β2+3 antibody 1:500 (Bioss, bs-12066R).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence