Journal: The Journal of Biological Chemistry

Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

doi: 10.1074/jbc.RA117.000262

Figure Lengend Snippet: Isolation of MIP from phage-displayed combinatorial peptide library. Following three rounds of affinity selection with a phage-displayed combinatorial peptide library, ANL7 , a single peptide ligand, MIP (NH 2 -AIRINPNGTWSRQAETVES-COOH; insert (underlined) and flanking region), was identified to bind a GST-MLK3 SH3 domain fusion protein. ELISA shows the phage-displayed MIP to bind to the WT form of the MLK3 SH3 domain (SH3 WT) but not the GST fusion partner. The presence of virions in the supernatant was determined by coating the wells of the microtiter plate with an anti-M13 mAb ( anti-M13 Ab ). Virions, which were retained in the microtiter plate wells, were detected with anti-M13 mAb conjugated to HRP. Experiments were performed in duplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S1 .

Article Snippet: After three washes with PBS containing 0.1% Tween 20 (PBST), retention of the SH3 domain fusion protein in wells was detected with an anti-GST antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare; 45 min; 1:5000 in PBST).

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

doi: 10.1074/jbc.RA117.000262

Figure Lengend Snippet: Binding properties of synthetic MIP. To determine the IC 50 value of MIP, a GST-MLK3 SH3(43–104) domain fusion protein was preincubated with increasing concentration of unlabeled MIP (AIRINPNGTWSRQAETVES) as competitor and then allowed to interact with biotinylated MIP (AIRINPNGTWSRQAETVES-K-biotin) immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP, and the levels are presented as percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. The curve fitting was performed with GraphPad Prism 6.0 software.

Article Snippet: After three washes with PBS containing 0.1% Tween 20 (PBST), retention of the SH3 domain fusion protein in wells was detected with an anti-GST antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare; 45 min; 1:5000 in PBST).

Techniques: Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Software

Journal: The Journal of Biological Chemistry

Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

doi: 10.1074/jbc.RA117.000262

Figure Lengend Snippet: Competition ELISA between MIP and NS5A peptide. To determine whether the MIP and NS5A peptides competitively bind the SH3 domain of MLK3, a GST-MLK3 SH3(43–104) fusion protein was preincubated with increasing concentrations of unlabeled MIP peptide or unlabeled NS5A peptide as competitor and then allowed to interact with biotinylated MIP ( A ) or biotinylated NS5A ( B ) peptide probes immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of the SH3 domain was detected with an anti-GST antibody conjugated to HRP, and the signal levels are presented as a percentage of binding in the absence of competitor. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. Curve fitting was performed with OriginPro 2017.

Article Snippet: After three washes with PBS containing 0.1% Tween 20 (PBST), retention of the SH3 domain fusion protein in wells was detected with an anti-GST antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare; 45 min; 1:5000 in PBST).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

doi: 10.1074/jbc.RA117.000262

Figure Lengend Snippet: Binding of MIP and NS5A peptides to SH3 domain of other members of MLK subfamily (MLK1–4). To determine whether MIP and NS5A can interact with SH3 domains of MLK1–4 proteins, the purified GST-MLK1–4 SH3 domains were incubated with biotinylated peptides, NS5A peptide (KKAPTPPPRRRR-GGG-K-biotin), MIP (AIRINPNGTWSRQAETVES-K-biotin), and MIP-R12A (AIRINPNGTWSAQAETVES-K-biotin), immobilized on a NeutrAvidin-coated 96-well ELISA plate. Binding of SH3 domain was detected with anti-GST antibody conjugated to HRP. Experiments were performed in triplicate, and the results are an averaged value; error bars reflect the standard deviation of each point. See also Fig. S6 .

Article Snippet: After three washes with PBS containing 0.1% Tween 20 (PBST), retention of the SH3 domain fusion protein in wells was detected with an anti-GST antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare; 45 min; 1:5000 in PBST).

Techniques: Binding Assay, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: iScience

Article Title: Metabolic rewiring and autophagy inhibition correct lysosomal storage disease in mucopolysaccharidosis IIIB

doi: 10.1016/j.isci.2024.108959

Figure Lengend Snippet:

Article Snippet: goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) , Santa Cruz Biotechnology , Cat# sc-2031; RRID: AB_631737.

Techniques: Recombinant, Modification, Protease Inhibitor, Expressing, Clone Assay, Control, Generated, Knock-Out, Software

Journal: International Journal of Molecular Sciences

Article Title: High Expression of the Lysosomal Protease Cathepsin D Confers Better Prognosis in Neuroblastoma Patients by Contrasting EGF-Induced Neuroblastoma Cell Growth

doi: 10.3390/ijms23094782

Figure Lengend Snippet: SH-SY5Y KD-CD cells are more sensitive to EGF and show a faster growth compared to CD-overexpressing cells. Assessment of cell proliferation following 20 ng/mL EGF treatment. ( A ) The figure shows a graphical representation of cell count, performed in triplicate for each experimental condition. The treatment was repeated every 24 h, until the end point of 72 h. Time zero is referring to the first day of treatment. ( B ) Immunofluorescence double staining at 24, 48, 72 h. Cells were stained for Ki-67 (green)/p21 (red). Scale bar = 20 μm; magnification = 63X. Representative images of different fields for each experimental condition are shown. ( C ) Cell cycle analysis performed on SH-SY5Y clones after 72 h of EGF. The percentage of cell populations in different cell cycle phases is reported. Quantification was performed by Flowing software 2.0.

Article Snippet: The following primary antibodies were employed for immunofluorescence: mouse anti-p21 (1:100, cod. sc-817; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-Ki-67 (1:100, cod.

Techniques: Cell Counting, Immunofluorescence, Double Staining, Staining, Cell Cycle Assay, Clone Assay, Software

Journal: Neuron

Article Title: Dopamine Triggers the Maturation of Striatal Spiny Projection Neuron Excitability during a Critical Period

doi: 10.1016/j.neuron.2018.06.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Tyrosine Hydroxylase monoclonal Millipore Cat # MAB5280 Rabbit anti-Red fluorescent protein polyclonal Rockland Cat # 600-401-379 Rabbit anti-DARPP32 monoclonal Cell Signaling Technology Cat # 2306S Mouse anti-beta actin monoclonal Novus Biologicals Cat # NB600-501 Mouse anti-Kir2.1 monoclonal Antibodies Incorporated Item # 73-210; RRID: AB_11000720 Mouse anti-Kir2.3 monoclonal Antibodies Incorporated Item # 75-069; RRID: AB_2130742 Mouse anti-K v 1.2 monoclonal Antibodies Incorporated Item # 75-008; RRID: AB_2296313 Rabbit anti- Phospho-(Ser/Thr) PKA Substrate Antibody polyclonal Cell Signaling Technology Cat # 9621S Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa 488 Invitrogen Cat # A-21202 Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa 594 Invitrogen Cat # A-21207 Donkey anti-Rabbit IgG (H+L) Secondary IRDye 680LT LI-COR P/N 925-68023 Goat anti-Mouse IgG (H+L) Secondary IRDye 800CW LI-COR P/N 925-32210 Streptavidin, Alexa 488 conjugate Invitrogen {"type":"entrez-protein","attrs":{"text":"S11223","term_id":"112468","term_text":"pir||S11223"}} S11223 Donkey anti-Mouse IgG (H+L) conjugated to HRP Jackson ImmunoResearch Code: 715-035-151 Chemicals, Peptides, and Recombinant Proteins Tetrodotoxin citrate Tocris Cat # 1069 Forskolin Tocris Cat # 1099/10 diC8-PIP2 Echelon Biosciences P-4508 Picrotoxin Tocris Cat # 1128/1G D-AP5 Tocris Cat # 0106 G418 disulfate salt (Neomycin) Tocris Cat # 4131/100 Neurobiotin tracer Vector Laboratories Cat # SP-1120 CNQX disodium salt Tocris Cat # 1045/1 Critical Commercial Assays BCA Protein Assay Kit Thermo Fisher Scientific Prod # 23227 Immobilon Western Chemiluminescent HRP Substrate Millipore Cat # WBKLS0500 Experimental Models: Organisms/Strains Mouse: Pitx3ak/2J The Jackson Laboratory Stock No: 000942 Mouse: B6.Cg-Tg(Drd1a-tdTomato)6Calak/J The Jackson Laboratory RRID: IMSR_JAX:016204 Mouse: WT: C57BL/6J The Jackson Laboratory RRID: IMSR_JAX:000664 Mouse: DAT KO Courtesy of Marc Caron Giros et al., 1996 Software and Algorithms GraphPad Prism GraphPad RRID: SCR_002798 Igor Wavemetrics RRID: SCR_000325 pClamp Molecular Devices RRID: SCR_011323 Image Studio Lite LI-COR RRID: SCR_014211 ImageJ NIH RRID: SCR_003070 Open in a separate window KEY RESOURCES TABLE Striatal dopamine release and projection neuron excitability mature postnatally Dopamine is required for the maturation of direct pathway SPN intrinsic excitability Maturation of direct pathway SPN excitability arises from PIP 2 -Kir2 interactions Dopamine must act during a critical period in development for this maturation

Techniques: Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Western Blot, Software