Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 1. Proteins (EGF, EGFR, and Grb2), EGFR component states, and protein−protein interfaces considered in our computational model. The EGFR ectodomain is taken to be free or bound to EGF. A cytoplasmic domain of EGFR, comprising the juxtamembrane region (JM) and kinase domain, is taken to be locked (i.e., unavailable for interaction) or freed (i.e., available for interaction). The C-terminal tail of EGFR is taken to contain, as a simplification, a single docking site for Grb2, which can be unphosphorylated (Y) and inactive or phosphorylated (pY) and active.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques:

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 2. Illustration of the rules for interactions in our computational, rule-based model. The model, which captures the mass-action chemical kinetics of the indicated interactions, consists of 16 rules, which are either reversible (and associated with two rate constants) or unidirectional (and associated with a single rate constant). Each rule represents an interaction. The glyphs used here to represent proteins and protein components are the same as those presented in Figure 1. Here, in illustrating a rule, we use a question mark (?) to indicate a missing protein component or component state that is not depicted explicitly; the missing component or state is taken to have zero influence on the interaction represented by the rule. Similarly, representation of an EGFR ectodomain by a dotted triangle is meant to indicate that the ectodomain may or may not be present in a complex, without influence on the interaction of concern. The model is the same as that presented in our earlier report25 except that a rule for Grb2 binding to phosphorylated EGFR has been added. This rule is illustrated in the lower left box.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 4. FLIM data of individual living BaF/3 cells represented on a phasor diagram. Data points correspond to BaF/3 cells transfected with EGFR−eGFP alone (blue diamond), EGFR−eGFP + EGF (second blue diamond), EGFR−eGFP/Grb2−mRFP (red-filled diamond), EGFR−eGFP/Grb2−mRFP + EGF (red-filled triangle), and untransfected control cells (blue filled circle). Blue solid line denotes trajectory for mixtures of background and EGFR−eGFP. Red line indicates trajectory for EGFR−eGFP/Grb2−mRFP FRET complex mixing with background and EGFR−eGFP fluorescence.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Transfection, Control

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 3. FLIM data of living BaF/3 cell populations represented on a phasor diagram. (A) Phasor diagram over a limited data range. (B) Phasor diagram on an expanded scale. Individual data points represent the cell-phasor components [x = m cos(φ); y = m sin(φ)] averaged from >20 cells. Data points correspond to BaF/3 cells transfected with EGFR−eGFP alone (blue diamond), EGFR−eGFP + EGF (second blue diamond), EGFR−eGFP/Grb2−mRFP (red-filled diamond), EGFR−eGFP/Grb2−mRFP + EGF (red-filled triangle), and un- transfected control cells (blue filled circle).

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Transfection, Control

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 5. FRET−FLIM−ICS on living BaF/3 cells cotransfected with EGFR−eGFP and Grb2−mRFP. (A) Fluorescence image of EGFR− eGFP/Grb2−mRFP complexes. (B) Spatial autocorrelation image of EGFR−eGFP/Grb2−mRFP complexes. (C) Density of Grb2−mRFP- bound EGFR−EGFP clusters as a function of the density of Grb2− free EGFR−eGFP clusters. The solid line is fit to a Hill function (CDbound = A/(1 + ((Kd/CDfree)(N−1)), with N = 4.1, A = 27, and Kd = 18 clusters).

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Fluorescence

Journal: Biochemistry

Article Title: Recruitment of the adaptor protein Grb2 to EGFR tetramers.

doi: 10.1021/bi500182x

Figure Lengend Snippet: Figure 6. (A) Plot of simulation results depicting the cluster distribution of Grb2-bound EGFR as a function of EGF concentration. Note that at all concentrations of EGF the EGFR tetramer is the predominant form associated with Grb2. The curves corresponding to dimer and trimer are indistinguishable from monomer because the total number of these oligomeric forms bound to Grb2 is almost negligible. (B) Cluster size distribution of EGFR bound to Grb2 and unbound (free) to Grb2 from simulation with 10 nM EGF.

Article Snippet: Human Grb2 (growth factor receptor-bound protein-2) transcript variant 1, as 10 μg of transfection-ready DNA, was purchased from OriGene (catalog no. SC111933) in the vector pCMV6-XL5.

Techniques: Concentration Assay

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 1. GRB2 complexes with AGO2 under non-stimulated conditions. Schematic diagram of, (a) AGO2 and, (b) GRB2 domain structures. Domains are named and colour coded and attributed amino acid sequence number. Red arrows indicate positions of PXXP motifs investigated in this work. (c) Western blot of AGO2 co-immunoprecipitated with GRB2 in serum starved HEK293T, A498 and PC3 cells. A longer exposure was used to capture AGO2 bands than for GRB2 and GAPDH. All images are taken from the same western blot. (d) Fluorescence and fluorescence resonance energy transfer signals of RFP-tagged GRB2 and GFP-tagged AGO2. HEK293T cells overexpressing fluorescent proteins were serum-starved before imaging. N = 3. Scale bars are 10 μm.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Sequencing, Western Blot, Immunoprecipitation, Fluorescence, Förster Resonance Energy Transfer, Imaging

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 2. Binding of GRB2 to AGO2 is mediated by GRB2 NSH3 and a PXXP motif in AGO2 PAZ domain. (a) Isothermal titration calorimetry (ITC) of a peptide spanning the proline-rich motif 323PHLP326 in AGO2 PAZ domain. (KD = 4.27 ± 1.17 µM). (b, c) ITC of MBP-tagged AGO2 PAZ domain titrated into GRB2. (b) PAZ WT (KD = 585 ± 61 nM). (c) No binding observed for mutation of PXXP (MBP-PAZ 4A). N = 2. (d) Fluorescence resonance energy transfer (FRET) between wild type (WT) and 323AAAA326 (4A) mutant GFP-tagged AGO2 and RFP-tagged GRB2 in HEK293T cells under conditions of serum starvation. White arrows indicate intracellular puncta which show increased FRET when WT AGO2 is expressed. N = 2. Scale bars are 10 μm. (e) Fluorescence lifetime imaging microscopy of RFP-tagged GRB2 proteins and GFP-AGO2 overexpressed in serum-starved HEK293T cells. The formation of a protein complex results in a reduction in fluorescent lifetime represented by a shift to the left of the population of fluorophores (measured in number of pixels). Lifetime population distribution shown by red line on graphs. x = Lifetime (ns), y = number of pixels. Solid black line corresponds to average fluorescent lifetime for GFP, 2.1 ns. Scale bars 25 μm. (f) Expanded region of interest (ROI) further exemplifying left-shift for AGO2/NSH3-SH2 interaction.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Mutagenesis, Fluorescence, Förster Resonance Energy Transfer, Imaging, Microscopy

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 3. Impact of GRB2-AGO2 complex on interaction with DICER1 and miRNA. (a) Western blot of AGO2 and DICER1 pulldown by GST-GRB2 in HEK293T cells. HEK293T cells were serum-starved before lysis. Bands captured with both a long and short exposure are shown for DICER1, whereas only the image captured with a short exposure is shown for AGO2. GST proteins were detected by ponceau stain. All images are taken from the same western blot. N = 3. (b–d) MST of AGO2 binding to DICER1 C-terminal region, upon pre-incubation of AGO2 with increasing concentrations of GRB2. The difference in binding affinity was negligible. (e) MST of GRB2 with DICER1 C-terminal region. No binding is observed within a physiologically relevant range hence the two do not interact directly. (f) Expanded ribbon model of molecular docking of GRB2 (green; PDB: 1GRI77) to AGO2 PAZ domain (cyan; red and blue indicate positive and negative charges respectively; PDB: 6RA478). The 323PHLP326 sequence is shown (yellow). GRB2 W36 (magenta) interacts with AGO2 P249 (red). Other residues in GRB2 which may contribute towards the interaction are shown in orange. Also shown is space-filling representation of AGO2 PAZ domain with PRM shown (below); and ribbon model of PAZ domain rotated by 90° to highlight juxtaposition of GRB2 binding site PRM and docking site for miRNA (right). Figures generated using PyMOL.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Western Blot, Lysis, Staining, Binding Assay, Incubation, Sequencing, Generated

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 4. GRB2 regulates miRNA expression in HEK293T cells. (a) Western blot of GRB2 expression in wild type (293 T) and depleted (G1) HEK293T clones 1 (G1.1) and 2 (G1.2). While G1.1 is a complete knockout, G1.2 contains a deletion and large insertion in the N-terminal SH3 domain. GRB2 was blotted with an antibody which recognised the C-terminal SH3 domain. Both long and short exposures were used to capture the GRB2 bands, whereas the GAPDH image was captured using a short exposure only. All images are taken from the same western blot. N = 3. (b) Heat plot highlighting miRNAs which show significant log2(fold changes) in expression (p < 0.05) between wild type HEK293T and G1 cells, measured by small RNA sequencing. Cells were deprived of growth factor. miRNAs demonstrated positive (red) and negative (blue) expression changes. N = 2. (c, d) RT-qPCR analysis of fold-change in mean expression of precursor miRNA transcripts (precursor and primary, pre-mir-, hashed bars) and mature miRNA (miR-, plain bars) derived from serum-starved G1 or wild type HEK293T cells. Two groups of miRNAs were observed: (c) miRNAs which diminished at both the level of the precursor and mature transcripts and, (d) miRNAs which were enhanced as mature transcripts but not as precursors. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 4. ns = not significant.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Western Blot, Clone Assay, Knock-Out, RNA Sequencing, Quantitative RT-PCR, Derivative Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Regulation of microRNA expression by the adaptor protein GRB2.

doi: 10.1038/s41598-023-36996-3

Figure Lengend Snippet: Figure 5. The GRB2-let-7 axis regulates oncogene expression. (a) RT-qPCR measurement of fold change in mean expression of let-7 g-5p miRNA and five target mRNAs in serum-starved GRB2 knockout cells (G1) compared to wild type HEK293T (293 T). Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3. (b) Western blot and (c) quantification of mean protein expression of let-7 targets in growth-factor-deprived G1 and HEK293T cells. The higher molecular band detected by the GRB2 antibody in G1 corresponds to an NSH3-mutated GRB2 polypeptide. For blot 1, a longer exposure was used to capture the DICER1 and GRB2 bands than was used for LIN28B and α-Tubulin. For blot 2, HMGA2 bands were captured using a longer exposure than that required for GRB2 and GAPDH. (d) Quantification of the area covered by migration of HEK293T cells expressing GFP-tagged wild type AGO2 (WT) or an AGO2 mutant which is incapable of binding GRB2 (4A), under conditions of reduced growth factor. Comparisons were made using a two-tailed Student’s t-test and error bars show standard error of mean. N = 3.

Article Snippet: GRB2 knockout was achieved using the homology-directed repair (HDR)-mediated knockout kit (OriGene, KN200469), which utilises CRISPR/Cas9 technology to insert puromycin resistance and GFP genes into the start of GRB2.

Techniques: Expressing, Quantitative RT-PCR, Knock-Out, Two Tailed Test, Western Blot, Migration, Mutagenesis, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: The Role of cAMP-dependent Signaling in Receptor-recognized Forms of α2-Macroglobulin-induced Cellular Proliferation

doi: 10.1074/jbc.m203543200

Figure Lengend Snippet: FIG. 9. Effect of 2M* and forskolin on the levels of c-Fos, Grb2, Sos 1/2, and Shc in macrophages. A, c-Fos; B, Grb2; C, Sos 1/2; D, Shc proteins in macrophages. The proteins were detected by Western blotting and quantified by a PhosphorImager as described under “Ex- perimental Procedures.” All panels, bar 1, buffer; bar 2, 2M* (100 pM); bar 3, fors- kolin (20 M); bar 4, forskolin and then 2M*. The corresponding gel blots are shown at the bottom of the respective bar graphs. The values are expressed in arbi- trary units and are the means S.E. from two or three independent experiments performed in triplicate.

Article Snippet: Antibodies against CREB, Rap-1, Raf-1, Raf-B, p70s6k, Grb2, Sos 1/2, and Shc were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Western Blot

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: bioRxiv

Article Title: Clathrin-coated Pits are Shredded Organelles Presorting Receptors in the Plasma Membrane

doi: 10.1101/491670

Figure Lengend Snippet: (A-J) TIRFM images of EGFP-CLC and JF549-labeled SNAP-adaptors (CLC, Cav1, AP2μ2, FCHO, Epsin2, EPS15, EPS8, PICALM, HIP1R and GRB2) ( upper panels ) for the analysis of colocalization between CCPs and the clusters of each adaptor in a single living cell. The individual clusters of each adaptor objectively detected using the particle detection algorithm (magenta dots) are displayed ( lower panels ). (K) The quantitative analysis of the colocalization ratios of CCPs with the clusters of each adaptor. The colocalization ratio of CCPs with EGFRs is shown (a red dashed line). (L) A histogram for the colocalization ratio of CCPs with EGFR after the knock-down of the indicated adaptors. (M, N) Maps showing conditional probability of the CCP subsets containing PICALM (red dots), given the subset containing EGFR (blue circles) (M) or vice versa (N). (O) An independence test for the events between the CCP subsets containing EGFR and the indicated adaptors, respectively. Error bars denote s.e.m. at the single-cell level (n > 5). Scale bars, 5 μm. *p < 0.01 (Student’s t -test).

Article Snippet: The Halo-conjugated GRB2 and PICALM were constructed by subcloning ( ) Halo into the pcDNA3.1 vector (#V800-20, Invitrogen, Carlsbad, CA) to construct pcDNA3.1/Halo-His from pENTR4-Halo obtained from Eric Campeau (Addgene plasmid #29644) with the primers 5’-GCTCT AGAGGAGGGAT GGCAGAAAT CGGT ACT GGC and 5’-GATCCACCGGTGCCGGAAATCTCGAGCGTCGA and ( ) GRB2 obtained from Dominic Esposito (Addgene plasmid #70383) and PICALM, a kind gift from Jin-moo Lee, were subcloned into the C-terminal of the pcDNA3.1/Halo-His vector with the following primers: 5’-GGAATTCATGGAAGCCATCGCCAAATATGACTTCAAA and 5’-GCTCTAGAGACGTTCCGGTT CACGGGGGT for GRB2 and 5’-CGGGATCCATGTCTGGCCAGAGCCTGACG and 5’-ATAAGAATGCGGCCGCTTCATAAACTGTATCTGTGCTCC for PICALM.

Techniques: Labeling

Journal: Journal of ovarian research

Article Title: Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

doi: 10.1186/s13048-023-01152-y

Figure Lengend Snippet: Fig. 1 The expressions of Gab2 and CrkII in ovarian cancer were detected by immunohistochemistry. A Typical diagram of Gab2 immunohistochemical results (above: ovarian cancer tissue, below: adjacent tissues). B Expression of Gab2 in ovarian cancer tissue and adjacent tissues. C Typical diagram of CrkII immunohistochemical results (above: ovarian cancer tissue, below: adjacent tissues). D Expression of CrkII in ovarian cancer tissue and adjacent tissues. E The relationship between Gab2 and CrkII. The X-axis and Y-axis represent the comprehensive score of Gab2 and CrkII expression in ovarian cancer tissues, respectively

Article Snippet: The primary antibodies of Gab2 and CrkII were diluted at 1:1000,GAPDH (Proteintech, 60004-1-Ig) was diluted at 1:2000 and the secondary antibodies, Mouse (Absin, abs20039) and Rabbit (Absin, abs20040), were diluted at 1:2000.

Techniques: Immunohistochemistry, Immunohistochemical staining, Expressing

Journal: Journal of ovarian research

Article Title: Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

doi: 10.1186/s13048-023-01152-y

Figure Lengend Snippet: Fig. 3 Expression of Gab2 and CrkII in ovarian cancer cell lines. A Western Blot was used to analysis of Gab2 and CrkII expression in ovarian cancer cell lines (A2780, SKOV3 and HO8910) and ovarian surface epithelial cells (IOSE80). B The expression of Gab2 in A2780 and SKOV3 transfected with Gab2 siRNA was elevated. C Evaluating the expression level of Gab2 in SKOV3 and A2780 cells were transfected with Gab2 overexpression virus. D The expression of CrkII in A2780 and SKOV3 transfected with CrkII knockdown virus was tested. E Testing the expression of CrkII in A2780 and SKOV3 were transfected with CrkII overexpression plastid

Article Snippet: The primary antibodies of Gab2 and CrkII were diluted at 1:1000,GAPDH (Proteintech, 60004-1-Ig) was diluted at 1:2000 and the secondary antibodies, Mouse (Absin, abs20039) and Rabbit (Absin, abs20040), were diluted at 1:2000.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Virus, Knockdown

Journal: Journal of ovarian research

Article Title: Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

doi: 10.1186/s13048-023-01152-y

Figure Lengend Snippet: Fig. 2 Relationship between Gab2 and CrkII and survival rate of patients with ovarian cancer. A Relationship between Gab2 and OS in patients. B Relationship between Gab2 and PFS in patients. C Relationship between CrkII and OS in patients. D Relationship between CrkII and PFS in patients

Article Snippet: The primary antibodies of Gab2 and CrkII were diluted at 1:1000,GAPDH (Proteintech, 60004-1-Ig) was diluted at 1:2000 and the secondary antibodies, Mouse (Absin, abs20039) and Rabbit (Absin, abs20040), were diluted at 1:2000.

Techniques:

Journal: Journal of ovarian research

Article Title: Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

doi: 10.1186/s13048-023-01152-y

Figure Lengend Snippet: Fig. 4 Effect of Gab2 on the biological behaviour of ovarian cancer cells. A Gab2 affects the migration ability of ovarian cancer cells. B Gab2 affects the proliferation of ovarian cancer cells. C Gab2 affects the sensitivity of ovarian cancer cells to carboplatin

Article Snippet: The primary antibodies of Gab2 and CrkII were diluted at 1:1000,GAPDH (Proteintech, 60004-1-Ig) was diluted at 1:2000 and the secondary antibodies, Mouse (Absin, abs20039) and Rabbit (Absin, abs20040), were diluted at 1:2000.

Techniques: Migration

Journal: Journal of ovarian research

Article Title: Gab2 plays a carcinogenic role in ovarian cancer by regulating CrkII.

doi: 10.1186/s13048-023-01152-y

Figure Lengend Snippet: Fig. 6 Gab2 affects the biological function of ovarian cancer cells through CrkII. A Co-IP was employed to determine the relationship between Gab2 and CrkII in A2780 and SKOV3. B Knockdown of CrkII reversed the migration of ovarian cancer cells induced by Gab2 overexpression. C Knockdown of CrkII reversed the proliferation of ovarian cancer cells induced by Gab2 overexpression. D The effect of Gab2 on the chemosensitivity of ovarian cancer cells depends on CrkII

Article Snippet: The primary antibodies of Gab2 and CrkII were diluted at 1:1000,GAPDH (Proteintech, 60004-1-Ig) was diluted at 1:2000 and the secondary antibodies, Mouse (Absin, abs20039) and Rabbit (Absin, abs20040), were diluted at 1:2000.

Techniques: Co-Immunoprecipitation Assay, Knockdown, Migration, Over Expression

Journal: Journal of Virology

Article Title: The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

doi: 10.1128/JVI.01464-18

Figure Lengend Snippet: 11-kDa specifically interacts with Grb2 but not P85α in vitro. (A to C) GST pulldown assay. Purified GST and GST–11-kDa were used as bait to pull down Grb2 from bacteria lysate. Numbered bands are as follows: 1, GST control; 2, Grb2; 3, GST–11-kDa; 4, putative Grb2 dimer; 5, putative GST–11-kDa dimer (A). Purified GST and GST–11-kDa were used as bait to pull down PI3K p85α and p85β subunits from bacterial lysate. Arrow shows the expressed p85α and p85β at ∼85 kDa (B). Purified GST and GST–11-kDa were used as bait to pull down Grb2 from lysate of M20-transfected UT7/Epo-S1 (S1) cells or B19V-infected EPCs. Both Coomassie blue staining and Western blotting were performed to examine the pulldown results (C). Diamond, Grb2; star, GST-11kDa monomers and dimers. (D and E) Biolayer interferometry (BLI) analysis of the interaction between 11-kDa and Grb2. BLI sensograms show the association and dissociation of Grb2 protein (at 2 μM) with WT 11-kDa at different concentrations, as indicated. Concentrations of 5 μM GST–11-kDa with 2 μM MBP-His and 5 μM GST with 2 μM MBP-Grb2-His were set up as negative controls (D). Binding parameters and Kdiss and Kass were used to calculate KD values (ratio of dissociation to association rate constant). Experiments were repeated at least three times for calculating the means and standard deviations of the KD.

Article Snippet: The following antibodies were purchased from the indicated vendors: anti-VP1/2 (MAB8293, clone R92-F6) from Millipore, rabbit anti-Grb2 (NB110-57013) from Novus, anti-pERK1/2 (Thr202/Tyr204) monoclonal antibody (4377) from Cell Signaling, and anti-β-actin (A5441) from Sigma.

Techniques: In Vitro, GST Pulldown Assay, Purification, Transfection, Infection, Staining, Western Blot, Binding Assay

Journal: Journal of Virology

Article Title: The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

doi: 10.1128/JVI.01464-18

Figure Lengend Snippet: 11-kDa specifically interacts with cellular Grb2 in vivo during infection. (A) Coimmunoprecipitation (co-IP) assay. An anti-Grb2 antibody was used to bind with and cross-link to beads, followed by pulldown of 11-kDa from lysate of B19V-infected EPCs at 48 h postinfection. Western blotting (WB) was used to identify the pulled down proteins, 11-kDa and Grb2, using anti-11-kDa and anti-Grb2, respectively. (B) Immunofluorescence assay. At 48 h postinfection, B19V-infected EPCs were harvested and coimmunostained for 11-kDa (red), Grb2 (green), and nucleus (blue) simultaneously. The cells were then visualized by confocal microscopy. DAPI, 4′,6′-diamidino-2-phenylindole.

Article Snippet: The following antibodies were purchased from the indicated vendors: anti-VP1/2 (MAB8293, clone R92-F6) from Millipore, rabbit anti-Grb2 (NB110-57013) from Novus, anti-pERK1/2 (Thr202/Tyr204) monoclonal antibody (4377) from Cell Signaling, and anti-β-actin (A5441) from Sigma.

Techniques: In Vivo, Infection, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: Journal of Virology

Article Title: The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

doi: 10.1128/JVI.01464-18

Figure Lengend Snippet: Grb2 knockdown facilitates B19V infection. (A and B) CD36+ EPCs on day 7 were transduced with lentiviruses. Cells were harvested at 48 h postransduction. The cells were analyzed by flow cytometry for cellular Grb2 and phosphorylated ERK (pERK) expression as values of mean fluorescence intensity (MFI). A representative experiment is shown in panel A. Statistical analyses of three independent experiments are plotted with levels relative to those of control cells. (C and D) CD36+ EPCs on day 7 were transduced with lentivirus expressing a scrambled shRNA (Scr.shRNA) or shRNA targeting Grb2 (Grb2shRNA), followed by B19V infection on day 9. Cells were harvested at 48 h postinfection and analyzed for NS1 expression as a percentage of NS1-expressing cells. A representative experiment is shown in panel C. Statistical analyses of NS1 expression levels from three independent experiments are plotted with levels relative to those of the Scr.shRNA-treated group. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following antibodies were purchased from the indicated vendors: anti-VP1/2 (MAB8293, clone R92-F6) from Millipore, rabbit anti-Grb2 (NB110-57013) from Novus, anti-pERK1/2 (Thr202/Tyr204) monoclonal antibody (4377) from Cell Signaling, and anti-β-actin (A5441) from Sigma.

Techniques: Infection, Transduction, Flow Cytometry, Expressing, Fluorescence, shRNA

Journal: Journal of Virology

Article Title: The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

doi: 10.1128/JVI.01464-18

Figure Lengend Snippet: One proline-rich motif is sufficient for 11-kDa to interact with Grb2 in vitro. (A) Mutations of proline-rich motifs. The proline (blue) residues which were mutated to alanine (red) in each mutant are shown. The numbers indicate the position of the amino acid residue in the 11-kDa protein. (B to E) Biolayer interferometry (BLI) BLI sensograms show association and dissociation of the MBP-Grb2-His protein (2 μM) with GST–11-kDa proline-rich (PR) motif mutants at different concentrations, as indicated. (F) Comparison of binding kinetics of all four PR mutants with that of WT 11-kDa. (G) Binding parameters. The KD value is the ratio of the dissociation to the association rate constant. Experiments were repeated at least three times for calculating the means and standard deviations of the KD.

Article Snippet: The following antibodies were purchased from the indicated vendors: anti-VP1/2 (MAB8293, clone R92-F6) from Millipore, rabbit anti-Grb2 (NB110-57013) from Novus, anti-pERK1/2 (Thr202/Tyr204) monoclonal antibody (4377) from Cell Signaling, and anti-β-actin (A5441) from Sigma.

Techniques: In Vitro, Mutagenesis, Binding Assay

Journal: Journal of Virology

Article Title: The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

doi: 10.1128/JVI.01464-18

Figure Lengend Snippet: Proposed model of the 11-kDa disruption of the EpoR-Grb2-SOS-Ras-MEK-ERK pathway. Upon Epo receptor (EpoR) activation through binding of Epo, Grb2 binds to tyrosine-phosphorylated EpoR either directly, through its SH2 domain, or indirectly, by binding to EpoR-associated tyrosine-phosphorylated SHC. Through the SH3 domain, Grb2 is constitutively associated with the guanine nucleotide-releasing factor (SOS) (29, 66, 67). Binding of Grb2 to EpoR leads to translocation of SOS to the membrane, where it triggers the exchange of GDP for GTP on Ras (68, 69); Ras-GTP then activates the Raf/MEK/ERK/ETS cascade (42). 11-kDa binding to Grb2 disrupts the interaction of Grb2 with SOS, which would block the exchange of GDP for GTP on Ras, and thereafter inhibits the Raf/MEK/ERK/ETS pathway. ERK activates ETS transcription factors, i.e., Elk-1 and c-Ets, which have been predicted to bind to the B19V replication origin (viral Ori) (20, 53).

Article Snippet: The following antibodies were purchased from the indicated vendors: anti-VP1/2 (MAB8293, clone R92-F6) from Millipore, rabbit anti-Grb2 (NB110-57013) from Novus, anti-pERK1/2 (Thr202/Tyr204) monoclonal antibody (4377) from Cell Signaling, and anti-β-actin (A5441) from Sigma.

Techniques: Activation Assay, Binding Assay, Translocation Assay, Blocking Assay