13771 1 ap  (Proteintech)

Journal: bioRxiv

Article Title: Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling

doi: 10.1101/2024.11.26.625481

Figure Lengend Snippet: (A) Proteins implicated in membrane trafficking SJ2BP and Syt4 colocalize with Cx36 in AII amacrine cells. (B) Several cytoskeleton associated proteins and regulators such as MAP6, DOCK7 or GPrin1 colocalize with Cx36 in AII cell dendrites. (C) The adhesion molecule BAI1 was often colocalized with Cx36 in AII cell dendrites. (D) Often components of chemical synapses such as SHANK2 and Glur2-3 were found in the periphery of gap junction plaques in AII amacrine cells. Scale: 10 µm. Magnified inset: 1 µm.

Article Snippet: The following primary antibodies were used: Cx36, 1:250 (Clone: 8F6, MAB3045, Millipore); Cx36, 1:250 (Clone: 1E5H5, 37-4600, Thermofisher); GFP, 1:250 (A10262, Thermofisher); ZO-1, 1:250 (Clone: ZO1-1A12, 33-9100, Thermofisher); ZO-2, 1:250 (71-1400, Thermofisher); CGN, 1:250 (PA5-5561, Thermofisher); SIPA1L3, 1:100 (30544-1-AP, Proteintech); EPS15L1, 1:250 (PA5-65940, Thermofisher); HIPR1, 1:250 (AB9882,Sigma); GluR2-3, 1:200 (07-598, Sigma); Gprin1, 1:100 (13771-1-AP, Proteintech); DOCK7, 1:100 (13000-1-AP, Proteintech); MAP6, 1:100 (NBP2-14220, Novus Biologicals); Synaptotagmin4, 1:100 (105 143, Synaptic Systems); SJ2BP, 1:100 (15666-1-AP, Proteintech); BAI1, 1:100 (NB110-81586, Novus Biologicals); SHANK2, 1:500 (Synaptic Systems, 162204); SCGN, 1:250 (CSB-PA020821LA01HU, ARP); SCGN, 1:500 (RD184120100, Biovendor); Vglut1, 1:500 (AB5905, Sigma); ITNS1, 1:100-250 (PA5-115432, Thermofisher).

Techniques: Membrane

Journal: Science signaling

Article Title: Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis.

doi: 10.1126/scisignal.aax9520

Figure Lengend Snippet: Fig. 1. Association of the 5-HT6R with GPRIN1 requires both the receptor C-terminal domain and Gs protein. (A) A simple interac- tion file was designed and imported in Cytos- cape (v2.8.1) to graphically show the interactions between the 5-HT6R and a network of proteins involved in the control of cytoskeletal dynamics. GPRIN1 is highlighted in pink. ERK, extracellular signal–regulated kinase; PALM, paralemmin; DOCK 3, dedicator of cytokinesis 3; WAVE1, WASP- family verprolin homologous protein 1; ARP2/3, actin-related protein 2/3; FYN, Fyn kinase. (B) Coimmunoprecipitation of the 5-HT6R and GPRIN1 from the brain extracts of newborn 5-HT6R–GFP KI mice. Western blots are represent ative of three independent experiments. IP, immunoprecipitation. (C) 5-HT6R immunostaining (assessed with an anti-GFP antibody, cyan) and GPRIN1 immunostaining (magenta) in the stria- tum from neonatal 5-HT6R–GFP KI mice. Scale bars, 5 m. a.u., arbitrary units. Right: Line graphs generated in ImageJ using the lines repre- sented on the merged image highlight the colocalization of both proteins at the plasma membrane in cell bodies (top), but not primary cilia (bottom). (D to H) NG108-15 cells were cotransfected with constructs encoding HA-tagged WT 5-HT6R or the indicated 5-HT6R mutants in the absence or presence of a GPRIN1 construct. (F and G) Cells were lysed 48 hours after trans- fection. (H) Cells were treated with 1 M SB271046 (SB; which was added 24 hours after transfec- tion and incubated for 24 hours), and the cells were lysed 48 hours after transfection. 5-HT6R pro- teins were immunoprecipitated with agarose- conjugated anti-HA antibodies. The presence of GPRIN1 (D and F to H) or Cdk5 (E) in the immunoprecipitated samples was assessed by Western blotting analysis. Western blots are representative of three independent experi- ments. Bar charts show densitometric analysis of the indicated bands and are means ± SEM of three experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to cells express- ing the 5-HT6R alone and exposed to vehicle. (I) BRET experiments measuring the interaction between the 5-HT6R–YFP and Cdk5-RLuc in NG108-15 cells 24 (top) and 48 hours (bottom) after transfection. Data are from four experiments.

Article Snippet: The agarose-conjugated antiHA antibody (clone no. HA-7) was purchased from Sigma-Aldrich, GFP-TRAP beads were from Chromotek, the rabbit anti-GPRIN1 antibody was from Proteintech (ref 13771-1-AP), the rabbit antiGs antibody was from Calbiochem (ref 371732), the rat anti-HA antibody (clone 3F10) was from Roche, the mouse anti-MAP2 (microtubule-associated protein 2) antibody (clone [HM2], ref ab11267) was from Abcam, and the mouse anti-Arl13b (clone N295B/66, ref 75-287) was from Antibodies Incorporated.

Techniques: Control, Western Blot, Immunoprecipitation, Immunostaining, Generated, Clinical Proteomics, Membrane, Construct, Incubation, Transfection

Journal: Science signaling

Article Title: Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis.

doi: 10.1126/scisignal.aax9520

Figure Lengend Snippet: Fig. 2. GPRIN1 increases 5-HT6R constitutive activity through Gs-cAMP signaling and promotes Gs binding to the receptor in NG108-15 cells. (A to C) The CAMY- EL cAMP signal was measured in NG108-15 cells transfected with constructs encoding the 5-HT6R (WT or W281A mutant) alone or in combination with a GPRIN1 construct 24 (A) or 48 hours (B and C) after transfection. Cells were treated with the TAT-Ct320–342 peptide (10 g, applied in the cell medium for 24 hours) or 1 M WAY181187 for 5 min (A), or the indicated concentrations of either WAY181187 or SB271046 (B) or vehicle (C) for 5 min. (B) Left: Data were normalized to the amount of cAMP in cells that did not express GPRIN1 and were challenged with 1 M WAY181187. Right: Data were normalized to the basal amount of cAMP in cells not expressing GPRIN1. Data are means ± SEM of values obtained from at least three independent experiments performed on different sets of cultured cells. **P < 0.01 and ***P < 0.001. (D and E) NG108- 15 cells were transfected with constructs encoding either HA-tagged WT or W281A 5-HT6R alone or in combination with the GPRIN1 construct and lysed 48 hours after transfection. (E) Cells were treated with control TAT peptide or TAT-Ct320–342 peptide (1 M, added 24 hours after transfection to the culture medium and incubated for 24 hours). 5-HT6R proteins were immunoprecipitated with agarose-conjugated anti-HA antibodies. The presence of GPRIN1 (D) or Gs (E) in the immunoprecipitated samples was assessed by Western blotting analysis. Western blots are representative of three independent experiments. Bar graphs show means ± SEM of immunoreactive signals measured in the three experiments. ***P < 0.001 compared to cells coexpressing WT 5-HT6R and GPRIN1 (D) and compared to cells coexpressing WT 5-HT6R and GPRIN1 and exposed to the control TAT peptide (E).

Article Snippet: The agarose-conjugated antiHA antibody (clone no. HA-7) was purchased from Sigma-Aldrich, GFP-TRAP beads were from Chromotek, the rabbit anti-GPRIN1 antibody was from Proteintech (ref 13771-1-AP), the rabbit antiGs antibody was from Calbiochem (ref 371732), the rat anti-HA antibody (clone 3F10) was from Roche, the mouse anti-MAP2 (microtubule-associated protein 2) antibody (clone [HM2], ref ab11267) was from Abcam, and the mouse anti-Arl13b (clone N295B/66, ref 75-287) was from Antibodies Incorporated.

Techniques: Activity Assay, Binding Assay, Transfection, Construct, Mutagenesis, Expressing, Cell Culture, Control, Incubation, Immunoprecipitation, Western Blot

Journal: Science signaling

Article Title: Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis.

doi: 10.1126/scisignal.aax9520

Figure Lengend Snippet: Fig. 3. GPRIN1 interaction with the 5-HT6R promotes neurite growth and branching in a PKA-dependent manner in NG108-15 cells. (A) NG108-15 cells were trans- fected with plasmids encoding GFP or YFP-tagged WT or W281A 5-HT6R alone or in combination with constructs encoding a dominant-negative Cdk5 mutant (Cdk5-DN; top) or GPRIN1 (bottom). GFP or receptor labeling is shown in cyan; GPRIN labeling is shown in magenta. Where indicated, cells were treated for 24 hours with the TAT- Ct320–342 peptide (1 M). The images show fields representative of three independent experiments performed on different cultures. Scale bar, 50 m. The graphs represent the branching index, measured as the number of intersections between dendrites and an overlaid concentric sphere, as a function of the distance from the soma. (B) Histogram showing the quantification of neurite length (m) in NG108-15 cells expressing the corresponding constructs and that were left untreated or were incubated with the TAT-Ct320–342 peptide. At least 300 neurites were analyzed per condition. ***P < 0.001. (C) Correlation plots between neurite length and basal cAMP amounts in NG108-15 measured either 24 (left) or 48 hours (right) after transfection of the cells with the indicated constructs. (D) NG108-15 cells were transfected with plasmids encoding YFP- tagged WT 5-HT6R or a mutant receptor incapable of coupling to G protein (Gs dead) alone or in combination with the GPRIN1 construct. Cells expressing the WT receptor were treated for 24 hours with 1 M SB271046, 25 M RPcAMP, or 2 M KT 5720. Cells were lysed 48 hours after transfection. Representative fields of three independent experiments performed on different sets of cultured cells are shown. Scale bar, 50 m. The graphs represent the branching index, which was measured as described in (A). (E) The histogram shows the quantification of neurite length in the indicated conditions, which were from the experiments shown in (D). ***P < 0.001.

Article Snippet: The agarose-conjugated antiHA antibody (clone no. HA-7) was purchased from Sigma-Aldrich, GFP-TRAP beads were from Chromotek, the rabbit anti-GPRIN1 antibody was from Proteintech (ref 13771-1-AP), the rabbit antiGs antibody was from Calbiochem (ref 371732), the rat anti-HA antibody (clone 3F10) was from Roche, the mouse anti-MAP2 (microtubule-associated protein 2) antibody (clone [HM2], ref ab11267) was from Abcam, and the mouse anti-Arl13b (clone N295B/66, ref 75-287) was from Antibodies Incorporated.

Techniques: Construct, Dominant Negative Mutation, Mutagenesis, Labeling, Expressing, Incubation, Transfection, Cell Culture

Journal: Science signaling

Article Title: Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis.

doi: 10.1126/scisignal.aax9520

Figure Lengend Snippet: Fig. 4. Interaction between endogenous 5-HT6R and GPRIN1 promotes neurite extension and the branching of striatal neurons. (A) Knockdown of GPRIN1 after infection of cultured mouse striatal neurons at DIV 4 by a virus expressing either control or two different GPRIN1-specific shRNAs. The neurons were lysed at DIV 13, and GPRIN1 was detected by Western blotting analysis. The bar graph shows the mean ± SEM of the GPRIN1 immunoreactive signal [normalized to the glyceraldehyde-3-phosphate dehydro- genase (GAPDH) immunoreactive signal] in each condition. ***P < 0.001 compared to neurons expressing the scrambled shRNA. (B) Neurons were infected with either control (top) or GPRIN1-specific (middle and bottom) shRNAs. Neurons were fixed at DIV 13. GPRIN1 immunoreactivity is shown in magenta, and MAP2 immunoreactivity in cyan. Scale bar, 5 m. The graph shows the measurement of neurite branching, as assessed by the Sholl analysis. The bar graph shows neurite length for neurons infected with control (black bar) or GPRIN1-specific shRNAs (white and gray bars). For both branching and neurite length, data are means ± SEM of three independent experiments performed from different sets of cultured neurons with at least 300 neu rites measured per conditions. ***P < 0.001. (C) Neurons were treated with either the con- trol TAT peptide (top) or the TAT-Ct320–342 peptide (1 M, added to cultures at DIV 4; bottom), and fixed at DIV 13. GPRIN1 is shown in magenta, and MAP2 in cyan. Neu- rite branching and length were determined and plotted as described in (B). *P < 0.05.

Article Snippet: The agarose-conjugated antiHA antibody (clone no. HA-7) was purchased from Sigma-Aldrich, GFP-TRAP beads were from Chromotek, the rabbit anti-GPRIN1 antibody was from Proteintech (ref 13771-1-AP), the rabbit antiGs antibody was from Calbiochem (ref 371732), the rat anti-HA antibody (clone 3F10) was from Roche, the mouse anti-MAP2 (microtubule-associated protein 2) antibody (clone [HM2], ref ab11267) was from Abcam, and the mouse anti-Arl13b (clone N295B/66, ref 75-287) was from Antibodies Incorporated.

Techniques: Knockdown, Infection, Cell Culture, Virus, Expressing, Control, Western Blot, shRNA

Journal: Science signaling

Article Title: Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis.

doi: 10.1126/scisignal.aax9520

Figure Lengend Snippet: Fig. 5. Schematic representation of the sequential interaction between the 5-HT6R and either Cdk5 or GPRIN1 and its consequence on agonist-independent 5-HT6R–stimulated signaling. Association of the 5-HT6R with Cdk5 results in receptor phosphorylation on a serine residue located in the receptor C-terminal (Cter) domain, a necessary step in the agonist-independent, receptor-mediated activation of Cdc42 signaling and initiation of neurite outgrowth (left), whereas the association of the receptor with GPRIN1 promotes the recruitment of activated Gs and neurite extension and branching through an agonist-independent, PKA-dependent mechanism (right). Nter, N-terminal. The application of an inverse agonist inhibits both pathways, indicating that these are dependent on the receptor’s constitutive activity.

Article Snippet: The agarose-conjugated antiHA antibody (clone no. HA-7) was purchased from Sigma-Aldrich, GFP-TRAP beads were from Chromotek, the rabbit anti-GPRIN1 antibody was from Proteintech (ref 13771-1-AP), the rabbit antiGs antibody was from Calbiochem (ref 371732), the rat anti-HA antibody (clone 3F10) was from Roche, the mouse anti-MAP2 (microtubule-associated protein 2) antibody (clone [HM2], ref ab11267) was from Abcam, and the mouse anti-Arl13b (clone N295B/66, ref 75-287) was from Antibodies Incorporated.

Techniques: Phospho-proteomics, Residue, Activation Assay, Activity Assay