Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Splenic mouse B cells deficient for Grb2 were retrovirally transfected with expression vectors encoding mouse γ2am (wt or YF) together with IRES-driven EGFP and Grb2 along with IRES-driven RFP. The resulting populations in the different gates (G1–G4) are shown on the right. The cells were stimulated with F(ab′) 2 fragments against IgG (α-IgG, indicated by an arrow, 20 μg ml −1 ) ( b – d ) or IgM (α-IgM, 20 μg ml −1 ) ( e ). ( b ) Ca 2+ mobilization kinetics of cells from all gates transfected with wild-type γ2am. ( c , d ) Ca 2+ mobilization kinetics of cells expressing either wild type (wt, blue curves) or YF-mutant (red curves) mIgG2a-BCRs from gate G4 ( c ) and G3 ( d ) on anti-IgG stimulation or G4 on anti-IgM stimulation ( e ). Ca 2+ mobilization was recorded in the presence of 1 mM extracellular CaCl 2 . Data are representative of three independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Transfection, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Experimental outline. Grb2 wt/wt Cγ1 cre/+ ( n =3) and Grb2 fl/fl Cγ1 cre/+ mice ( n =5) were repeatedly immunized with purified gB with aluminium hydroxide (alum) at the indicated time points. Splenic B cells were purified 70 days after third immunization by complement-mediated T-cell lysis and anti-CD19 magnetic bead separation. CD19-positive cells containing 800 memory B cells each (identified as B220+, IgG1+, gB+) were transferred intravenously into Rag1 −/− recipient mice and challenged 6 days later by an intravenous injection of 2 μg virus-like particles (VLPs) of human CMV in PBS. ( b ) gB-specific IgG1 titres were measured by ELISA. ( c ) ELISPOT for gB-specific IgG1-secreting cells was performed at day 43 after VLP challenge. Error bars represent mean+s.d. of at least three analyses; Student’s t -test was used. * P <0.05 ** P <0.01.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Purification, Lysis, Injection, Virus, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: ( a ) Schematic illustration of chimeric receptor variants used in this study. Chimeric constructs consisting of mouse YF-mutant γ2am fused to the N-terminal ( b ) or C-terminal ( c ) SH3 domains of Grb2 were retrovirally expressed in DG75 B cells. The SH3 domains were either wild type (wt, blue curves) or inactivated by tryptophan to lysine (W36K and W193K, respectively, red curves) or a phenylalanine to alanine (F165A, orange curve) mutations. Cells were either stimulated with anti-IgG or anti-IgM F(ab′) 2 fragments (indicated by arrows) and Ca 2+ mobilization was recorded in the presence of 1 mM extracellular CaCl 2 . ( d ) Chimeric γ2am-YF-chimeras containing the N-terminal SH3 domain of either Grb2 (wt: dark blue line, W36K: dark red line) or GRAP (wt: light blue line, W36K: light red line) were retrovirally expressed in murine splenic B cells and analysed as before. Data are representative of four independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Construct, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: DG75 B cells were stimulated via their BCR for 3 min to induce tyrosine phosphorylation of signalling proteins. Subsequently, lysates of these cells were used for affinity purifications with GST-coupled N-terminal SH3 domains of Grb2 and GRAP. Inactivated (W36K) variants were used as controls. Purified proteins were analysed by immunoblotting with anti-phosphotyrosine (α-p-Tyr) antibodies (upper panel) and antibodies to c-Cbl, Sos, SLP65 and Btk as indicated. To assess the efficiency of affinity purification, signals of lysates (equivalent to 2% of input lysates) are shown on the right. Data are representative of four independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Phospho-proteomics, Purification, Western Blot, Affinity Purification

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: BTK -deficient DG75 B cells were retrovirally transduced to express chimeric γ2am-YF molecules containing either the N-terminal (blue lines) or C-terminal (red lines) SH3 domains of Grb2. Subsequently, the cells were retrovirally transfected with Citrine-tagged Btk, resulting in two populations that were either Btk-negative (Cit-neg) or Btk-positive (Cit-pos) ( a ). BCR-induced Ca 2+ mobilization was analysed in all populations on stimulation with polyclonal F(ab′) 2 fragments to IgG ( b ) or IgM ( c ). ( d ) Wild-type DG75 cells expressing chimeric γ2am-YF molecules containing either the N-terminal SH3 domain of GRAP (red line) or a variant thereof (XIII) having three amino-acid substitutions at positions three, four and five (S3A, V4I and L6K, blue line) were analysed as before. ( e ) The same chimeric receptors as in ( d ) were analysed in BTK-deficient DG75 cells. ( f ) BTK -deficient DG75 cells were retrovirally transfected with wild type (wt, blue line) or tyrosine to alanine mutant (YA, red line) γ2am-encoding expression vectors and stimulated as before. Data are representative of at least three independent experiments.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Transfection, Expressing, Variant Assay, Mutagenesis

Journal: Nature Communications

Article Title: The immunoglobulin tail tyrosine motif upgrades memory-type BCRs by incorporating a Grb2-Btk signalling module

doi: 10.1038/ncomms6456

Figure Lengend Snippet: On phosphorylation by ITAM-bound Syk, the ITT motifs (white boxes in cytoplasmic mIgG tails) provide docking sites for the ubiquitous adaptor protein Grb2. Grb2 in turn brings along Bruton’s tyrosine kinase (Btk) via a constitutive interaction that is mediated by the N-terminal SH3 domain of Grb2. The incorporation of Grb2/Btk into the BCR signalosome stabilizes the Ca 2+ initiation complex consisting of Btk, the adaptor protein SLP65 (which in addition interacts with a non-ITAM tyrosine-phosphorylation motif in Igα) and phospholipase C-γ2 (PLC-γ2) at the activated receptor, and thereby lowers the threshold for activation of PLC-γ2 by Btk. This active signal amplification loop is complemented by a passive signal amplification that is brought about by sequestration of ITT-bound Grb2 from negative regulators of Ca 2+ mobilization such as CD22 and Dok-3.

Article Snippet: Splenocytes from 8- to 10-week-old female C57BL/6 mice (in house breeding) or corresponding B-cell-specific grb2 -ko ( Grb2 fl/fl mb1 cre/+ ) mice were depleted of CD43-positive cells using anti-CD43 magnetic microbeads (Miltenyi Biotec).

Techniques: Phospho-proteomics, Activation Assay, Amplification

Journal: BMB Reports

Article Title: YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR

doi: 10.5483/BMBRep.2014.47.10.151

Figure Lengend Snippet: Identification of an EGF-derived agonist peptide, Pep2-YAC. (A) The amino acid sequences covering residues 19-32 in EGF from 10 different species were aligned. The numbers indicate the position of a particular amino acid in the EGF. The underlined sequences represent conserved sequences used for the synthesis of agonist tripeptide candidates as follows: Pep1-VCM, Pep2-YAC, and Pep3-ACN. (B) In situ PLA reactions were performed to determine the agonistic effect of three peptides on the association of EGFR with Gab1 or Grb2 with SHP2 using specific antibodies. HaCaT cells were counterstained with DAPI (blue) to visualize nuclei. Red spots represent the interactions between the indicated proteins of interest. Scale bar, 5 μm. Original magnification, ×600. (C) Graph shows the effects of three EGF agonist peptide candidates on HaCaT cell proliferation, determined using the CCK-8 assay, as described in the Materials and Methods. The y-axis represents the absorbance values that were measured at a wavelength of 450 nm. **P ＜ 0.01, ***P ＜ 0.001, ****P ＜ 0.0001.

Article Snippet: Antibodies used for PLA were as follow: rabbit anti-EGFR Ab (Santa Cruz Biotechnology), mouse anti-Grb2 Ab (Santa Cruz Biotechnology), rabbit anti-Gab1 Ab (Cell signaling technology), mouse anti-SHP2 Ab (Santa Cruz Biotechnology), rabbit anti-Gab1 Ab (Cell Signaling Technology), mouse anti-PI3K p85 Ab (Santa Cruz Biotechnology), rab bit-c-jun (Santa Cruz Biotechnology), and mouse-c-fos (Santa Cruz Biotechnology).

Techniques: Derivative Assay, In Situ, CCK-8 Assay

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 1. Double immunofluorescence labeling, immunohistochemistry and Western blot analysis of the expression pattern of endophilin A1 in TLE patients. (A) Representative images of double immunofluorescence for endophilin A1 (green) and NeuN (red) or GFAP (red). Endophilin A1 and NeuN are coexpressed (merged), and endophilin A1 and GFAP are not coexpressed (merged) in the temporal neocortex of patients with TLE. Cellular nuclei are shown with DAPI staining (blue). Arrows show the positive cells (scale bar = 50 μm). (B) Representative images of immunohistochemical staining of endophilin A1-positive cells. Strong immunoreactive staining of endophilin A1 in the temporal neocortex is shown in TLE patients, in contrast to the faint staining in the control group. Arrows show the positive cells (scale bar = 50 μm). (C) Immunohistochemical analysis. The mean density value of endophilin A1 in TLE patients was significantly higher than that in the control group (*P < 0.05, unpaired t-test, n = 12 in TLE group and n = 11 in control group). (D) Representative images of endophilin A1 expression (37 kDa) in the human temporal neocortex. (E) Comparison of the intensity ratios in the Western blot indicates significantly higher expression of endophilin A1 in the epilepsy group than in the control group (*P < 0.05, unpaired t-test; humans: n = 10 in TLE group and n = 10 in control group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Labeling, Immunohistochemistry, Western Blot, Expressing, Staining, Immunohistochemical staining, Control, Comparison

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 3. Western blot and immunohistochemistry analysis of endophilin A1 in the PTZ-kindled epileptic mouse model. (A–B) Representative images (left) and statistical graphs (right) of endophilin A1 expression (37 kDa) in the hippocampus (A) and adjacent temporal cortex (B) of mice. Comparison of the intensity ratio of Western blot indicates significantly higher expression of endophilin A1 in the epilepsy group than that in the control group (*P < 0.05, unpaired t-test, n = 5). (C–D) Representative images (left) and statistical graphs (right) of immunohistochemical analysis of endophilin A1-positive cells. In the mouse hippocampus (C) and adjacent temporal cortex (D), strong immunoreactive staining of endophilin A1 appeared in the epilepsy group, in contrast to the faint staining in the control group. Arrows show the positive cells (scale bar = 50 μm). The mean density value of endophilin A1 in the epilepsy group was significantly higher than that in the control group (*P < 0.05, unpaired t-test, n = 5).

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Western Blot, Immunohistochemistry, Expressing, Comparison, Control, Immunohistochemical staining, Staining

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 4. Expression of endophilin A1 with green fluorescent protein (GFP) in the hippocampus after stereotaxic injection of recombinant LV. (A) Immunofluorescent image showing GFP expression (green) in the hippocampus after 14 days of stereotaxic injection of recombinant LV. Cellular nuclei are shown with DAPI staining (blue) (scale bar = 500 μm). (B) Western blotting images showing endophilin A1 expressions with or without injection of LV-GFP and LV-SH3GL2-RNAi on day 14, day 30 and day 45. (C) Comparison of mean intensity ratio shows significantly decreased expression of endophilin A1 in the LV-SH3GL2-RNAi group compared with other groups (*P < 0.05 versus control and LV-GFP. ANOVA test, n = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Expressing, Injection, Recombinant, Staining, Western Blot, Comparison, Control

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 5. Expression of endophilin A1 in the hippocampus of PTZ-kindled epileptic mouse model after stereotaxic injection of recombinant LV and its effects on PTZ kindling process. (A) Representative images of immunofluorescence for endophilin A1 (red) and LV-GFP (green). Endophilin A1 and recombinant LV are coexpressed. Cellular nuclei are shown with DAPI staining (blue). Low fluorescence intensity of endophilin A1 was observed in the hippocampus of the LV-SH3GL2-RNAi transduced epileptic mouse model (scale bar = 50 μm). (B) The mean intensity value of endophilin A1 (% of con) in the hippocampus of the LV-SH3GL2-RNAi transduced epilepsy group was significantly lower than that in the LV-GFP transduced epilepsy group (*P < 0.05, unpaired t-test, n = 5). (C) Western blotting images showing endophilin A1 expressions in LV-GFP and LV-SH3GL2-RNAi mice after PTZ kindled. (D) Comparison of the mean intensity ratios in the Western blot analysis of PTZ-kindled epileptic mice shows significantly decreased expression of endophilin A1 in the LV-SH3GL2-RNAi group compared with the LV-GFP group (*P < 0.05, unpaired t-test, n = 5). (E–G) The mean latency of first seizure onset was significantly increased (E) and the mean duration of Racine scale 4-5seizures (F) and number of Racine scale 4–5 seizures (G) were decreased in the LV-SH3GL2-RNAi group compared with the LV-GFP group (*P < 0.05, unpaired t-test, n = 9). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Expressing, Injection, Recombinant, Staining, Western Blot, Comparison

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 6. Electrophysiological changes in pyramidal neurons in the hippocampal CA3 area of brain slices after endophilin A1 knockdown. (A) Representative traces of AP in slices from LV- GFP-treated and LV-SH3GL2-RNAi-treated mice in Mg2+-free ACSF. (B) LV-SH3GL2-RNAi significantly decreased the average frequency of APs. (*P < 0.05, unpaired t-test, n = 5) (C) Sample traces of mEPSCs in each group. (D–E) LV-SH3GL2-RNAi treatment significantly decreased the average mEPSC amplitude (*P < 0.05, unpaired t-test, n = 5) (D), but not the average mEPSC frequency (*P > 0.05, unpaired t-test, n = 5) (E), compared with the LV-GFP group.

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Knockdown

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 8. Expression of intracellular and surface AMPAR GluR2 in LV-GFP and LV-SH3GL2-RNAi mice after PTZ kindling; co-immunoprecipitation of endophilin A1 and AMPAR GluR2. (A) Western blotting images showing intracellular and surface AMPAR GluR2 expression in LV-GFP and LV-SH3GL2-RNAi mice after PTZ kindled. (B) The intracellular expression of AMPA- GluR2 shows no significant difference between the groups (P > 0.05, unpaired t-test, n = 5). (C) Comparison of mean intensity ratios shows significantly decreased surface expression and surface/intracellular ratio of AMPAR GluR2 in the LV-SH3GL2-RNAi group compared with the LV-GFP group (*P < 0.05, unpaired t-test, n = 5). (D–E) Co-immunoprecipitation experiments of endophilin A1 and AMPAR GluR2 in the temporal neocortex of TLE patients (D) and the hippocampus of the PTZ-kindled epileptic mouse model (E), both showing that endophilin A1 interacted with AMPAR GluR2.

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Expressing, Immunoprecipitation, Western Blot, Comparison

Journal: Experimental neurology

Article Title: Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model.

doi: 10.1016/j.expneurol.2018.02.014

Figure Lengend Snippet: Fig. 10. Endophilin A1 knockdown fails to reduce seizure activity under the administration of the selective AMPAR agonist CX546. (A) Experimental design for investigating behavioral effects of endophilin A1 knockdown with or without CX546. Mice were injected bilaterally with LV-SH3GL2-RNAi in the hippocampus 2 weeks prior to daily intraperitoneal injection with either CX546 or vehicle, and PTZ was administered after 3 days. (B) Western blotting images showing intracellular and surface AMPAR GluR2 expression in LV-SH3GL2-RNAi+DMSO +PTZ group and LV-SH3GL2-RNAi+CX546+PTZ group. (C) The intracellular expression of AMPA-GluR2 shows no significant difference between the groups (P > 0.05, unpaired t-test, n = 5). (D) Comparison of mean intensity ratios shows significantly increased surface expression and surface/intracellular ratio of AMPAR GluR2 in the CX546 treated group compared with the vehicle treated group (*P < 0.05, unpaired t-test, n = 5). (E–G) The injection of the CX546 in endophilin A1 knockdown mice significantly decreased the latency of the first seizure (E) and increased the mean duration of Racine scale 4–5 seizures (F) and the total number of Racine scale 4–5 seizures (G) compared with the vehicle treated endophilin A1 knockdown mice (*P < 0.05, unpaired t-test, n = 9).

Article Snippet: The supernatants were collected and incubated with rabbit polyclonal antirabbit IgG antibody (2 μl; catalog number: ab171870; Abcam, USA), rabbit polyclonal anti-endophilin A1 antibody (4 μl; catalog number: 12435-1-AP; Proteintech, Wuhan, China), or rabbit monoclonal antiionotropic glutamate receptor 2 antibody (2 μl; catalog number: ab206293; Abcam, USA) overnight at 4 °C, followed by the addition of 40 μl of protein A+G agarose (catalog number: P2012; Beyotime, Shanghai, China) for 3 h at 4 °C.·Immunoprecipitates were collected and washed with lysis buffer 5 times after being centrifuged at 1000g for 5min and were then subjected to western blot analysis.

Techniques: Knockdown, Activity Assay, Injection, Western Blot, Expressing, Comparison

Journal: Science Advances

Article Title: Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein

doi: 10.1126/sciadv.adf5143

Figure Lengend Snippet: ( A ) Illustration of the domains of the proteins. Scale bar, 100 amino acid residues. ( B ) Binding of Nck, WISH, and GAS7 isoforms to N-WASP and its ΔPRR mutant. Nck, WISH, and GAS7 isoforms (1 μM) as GST fusion proteins were incubated with N-WASP and its ΔPRR mutant (0.5 μM). The bound proteins were analyzed by SDS–polyacrylamide gel electrophoresis, followed by Western blotting. GST was used as a negative control, and (−) indicates GST fusion protein alone. ( C and D ) Binding curve of immobilized GAS7b (0.1 μM) without GST to N-WASP (C) and WASP and its S339Y, P359T, and P373S mutants (D) by the biolayer interferometry analysis. The K d values ( n = 3) are shown on the right. The means ± SE are shown for the sensorgrams and K d values ( n = 3). ( E ) Binding of N-WASP and GAS7 isoforms to Nck and Grb2 (1 μM) as in (B). MW, molecular weight. ( F ) Binding of WISH and its Y52A mutant to splicing isoforms of GAS7 (0.5 μM) as in (B). ( G ) Binding curve of the immobilized Nck (0.1 μM) to WASP and its S339Y, P359T, and P373S mutants by the biolayer interferometry analysis, as in (D). ( H ) Binding of WISH and its Y52A mutant to N-WASP (0.5 μM) as in (B). ( I ) Binding of WISH and its Y52A mutant to Nck (0.5 μM) as in (B). ( J ) Binding curve of the immobilized WISH (0.1 μM) (H) to WASP and its S339Y, P359T, and P373S mutants by the biolayer interferometry analysis, as in (D). The P values were obtained using the one-way ANOVA with Dunnett’s post hoc analysis (D and G) and the Kruskal-Wallis test, followed by Dunn’s test (J). Significance values are * P < 0.05 and **** P < 0.0001. ns, not significant.

Article Snippet: The membranes were incubated with the primary antibody: mouse anti-GAS7 (clone 2F6, TA501756, OriGene), anti-Grb2 (Sc-8034, OriGene), anti-Nck (SC-20026, Santa Cruz Biotechnology), anti–N-WASP (#4848, Cell Signaling Technology), anti-WASP (#48606, Cell Signaling Technology), anti-SPIN90 (Ab88467, Abcam), anti-mCherry (71615, Cell Signaling Technology), and anti–glyceraldehyde-3-phosphate dehydrogenase (SC16657, Santa Cruz Biotechnology) at a 1:10,000 dilution, followed by an anti-mouse or anti-rabbit IgG alkaline phosphatase conjugate (Promega) secondary antibody in PBS-T at a 1:10,000 dilution.

Techniques: Binding Assay, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Negative Control, Molecular Weight