pgex2t plasmid  (Addgene inc)

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet:

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Virus, Plasmid Preparation, Recombinant, Mass Spectrometry, Knock-Out, Sequencing, Control

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet: (A) Coronal mouse brain section showing ipsilateral (Ipsi) hippocampus injected with shRNA AAVs and GFP and non-injected contralateral (Con) side; 3 weeks post-injection. Scale bar, 200 μm. (B)Western blots of dissected hippocampus 3 weeks after injections show shRNAs reduce Sam68 expression. Representative of 3 blots. (C)RNA IP from WT or Sam68 KO hippocampal lysates shows that Sam68 binds to Arc mRNA but not CaMKIIα or MAP2 mRNAs. Representative of 4 RNA IPs. (D) Magnified views of white boxes from (A) encompassing the s. pyramidale (within dashed lines) and s. radiatum layers of CA1 (right of s. pyramidale ). Shown is DAPI (nuclei, blue), GFP signal, and RNAscope imaging of Arc mRNA in injected (Ipsi) hemisphere (top panels) and uninjected (Con) hemisphere (bottom panels). Scale bar, 50 μm. (E) Arc mRNA distribution plotted as percent difference in localization (mRNA enrichment in Ipsi compared to the Con hemisphere) versus distance from the cell body; 0–50 μm = proximal, >50 μm = distal. Left panel, shS68 increased Arc mRNA levels in proximal regions and decreased levels in distal regions. No differences were observed with shNT (4 mice per condition; 5 slices per animal, shS68 Ipsi versus Con; Mann-Whitney test, U = 51, p < 0.05). Right panel, Sam68 knockdown had no effect on CaMKIIα mRNA distribution (4 mice per condition; 5 slices per animal, Mann-Whitney test, U = 93, p > 0.05). (F)Cumulative frequency distribution plots of data presented in (E), showing a significant difference (two sample Kolmogorov-Smirnov test, KS-test) for Arc mRNA localization (left panel, shS68 Ipsi versus Con, D = 0.124, p < 0.05; shNT Ipsi versus Con, D = 0.02, p > 0.05) but not CaMKIIα mRNA localization (right panel, shS68 Ipsi versus Con, D = 0.04, p > 0.05; shNT Ipsi versus Con, D = 0.04, p > 0.05). (G) Representative qRT-PCR for Arc mRNA in primary neurons transduced with shS68 or shNT lentiviral shRNAs and treated with the transcriptional inhibitor actinomycin D for the indicated time points (min). Below, qRT-PCR quantitation shows Sam68 knockdown does not affect Arc mRNA degradation. n = 5 biological replicates (Mann Whitney test, U = 15, p > 0.05). Data points represent mean ± SEM. (H) Co-immunoprecipitations from cortical brain lysates show Sam68 interacts with kinesin molecular motor KIF5A but not KIF1b or KIF17.

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Injection, shRNA, Western Blot, Expressing, RNAscope, Imaging, MANN-WHITNEY, Knockdown, Quantitative RT-PCR, Transduction, Quantitation Assay

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet: (A) Primary neuronal cultures infected with lentiviral shRNAs and imaged for Arc protein (gray) and MAP2 (magenta) in GFP-positive cells (GFP not shown). Scale bar, 10 μm. Below (left), Arc protein intensity quantified using ImageJ, normalized to levels in the soma (% Arc), and plotted as a function of distance from the cell body. Below (right), Sam68 knockdown significantly reduced Arc protein only in distal dendritic regions (>100 μm). n = 28 neurons; three independent experiments (Mann-Whitney, U = 1166, ***p < 0.0001). (B) Primary neurons infected as in (A) were treated with puromycin (10 μM) for 15 min and fixed. Loss of Sam68 does not affect global protein synthesis (quantified using anti-puromycin antibodies) proximal or distal to cell bodies. n = 13 neurons; three independent experiments. (C) Primary neurons were treated with cycloheximide to inhibit translation. Western blots show that loss of Sam68 had no significant effect on Arc protein half-life. n = 3 biological replicates. For (B) and (C), Mann-Whitney U tests give p values >0.05. (D) Rabbit-reticulocyte-based in vitro translation assay using Arc mRNA and increasing amounts of purified GST or GST-Sam68 protein. Western blots and quantitation below show that Sam68, but not GST, increases Arc translation. RPS3, control ribosomal marker. n = 5 biological replicates. Two-way ANOVA with Sidak post hoc for multiple comparisons, *p < 0.05, ***p < 0.0005. All box and whisker plots indicate mean, 25%–75% percentiles, and min to max range. Data points in (C) and bar graph in (D) represent mean ± SEM.

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Infection, Knockdown, MANN-WHITNEY, Western Blot, In Vitro, Purification, Quantitation Assay, Control, Marker, Whisker Assay

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet: (A) Field recordings following DHPG-induced mGluR-LTD (DHPG-LTD; 50 μM, 5 min) in acute hippocampal slices at proximal (~40 μm from cell body) and distal (~150 μm from cell body) Schaffer collateral synapses. WT mice show no significant difference between dendritic areas (proximal [33 slices; 9 mice]: distal [32 slices; 9 mice]). Right panel, Sam68 KO mice show significantly impaired DHPG-LTD at distal (17 slices; 4 mice) but not proximal synapses (17 slices; 4 mice). (B) HET Sam68 KO mice show similar deficits at distal (15 slices; 5 mice) but not proximal synapses (15 slices; 5 mice). (C) Left panel, WT mice show no significant difference in the magnitude of synaptically induced paired-pulse LFS-LTD (900 pulses; 1Hz; 15 min, 50-ms paired-pulse interval) between proximal inputs (8 slices; 4 mice) and distal inputs (8 slices; 4 mice). Right panel, In Sam68 HET mice, synaptically induced LTD was impaired at distal (8 slices; 4 mice) but not proximal synapses (8 slices; 4 mice). (D) Field recordings in acute hippocampal slices show that transection of the cell-body layer in Sam68 KO slices (KO cut; 10 cut slices; 4 mice) abolished the DHPG-LTD observed at proximal synapses in untransected slices (KO uncut; 8 control slices; 4 mice). The magnitude of LTD in transected slices from WT mice was comparable to uncut KO slices (WT cut; 4 slices from 2 mice). One-way ANOVA F(2,9) = 17.2, p < 0.05; paired comparisons; KO cut versus KO uncut, p < 0.05; KO cut versus WT cut, p < 0.05; KO uncut versus WT cut, p > 0.05). For all experiments, data plotted represent mean ± SEM, and paired representative traces displayed are for baseline and post-LTD; scale bar represents 0.25 mV, and 10 ms. The average % LTD (from pooled slices per animal) calculated across the last 5 minutes of recording were used for statistical evaluation. Two-tailed Student’s t test, *p < 0.05 and n = number of animals were used for (A), (B), and (C).

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Control, Two Tailed Test

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet: (A) IP and isobaric labeling process showing eluted immunocomplexes reacted with unique isobaric tags; 10 plex. (B) Western blots using 1/100 of the eluted complexes confirm Sam68 immunopurification. (C) Percent overlap between the Sam68 interactome (Sepharose) and other interactomes with statistics calculated using a hype rgeometric means distribution analyses (PDF). (D) Network analysis for proteins identified in the Sam68 interactome with at least 2 peptides identified and >2-fold enrichment over background. A total of 151 proteins were identified and ≥ medium confidence (0.4-String.db) interactions are displayed. A total of 245 interactions (edges) were identified compared to an expected 119 based on random chance. (E) Ontological analysis of the Sam68 interactome by using 1 detected peptide and at least 1.5-fold enrichment over background identified 534 proteins (see ). The top 5 for indicated classification are listed, showing a strong role for Sam68 in protein translation and RNA metabolism (see ).

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Labeling, Western Blot, Immu-Puri

Journal: Cell reports

Article Title: Sam68 Enables Metabotropic Glutamate Receptor-Dependent LTD in Distal Dendritic Regions of CA1 Hippocampal Neurons

doi: 10.1016/j.celrep.2019.10.030

Figure Lengend Snippet:

Article Snippet: Bacterial expression plasmid used to generate Sam68 protein is available at Addgene as pGEX-2T-Sam68 (# 17687).

Techniques: Virus, Plasmid Preparation, Recombinant, Mass Spectrometry, Knock-Out, Sequencing, Control

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left ; and IBRS2, right ) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 ( red ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 ( a ) or mouse monoclonal anti-G3BP ( b ) followed by donkey-anti-goat-AF568 ( red , a ) or goat-anti-mouse-AF568 ( red ; b ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section ( b ). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section ( b ) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing, Binding Assay, Western Blot, Labeling, Mutagenesis, Construct, Control

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Oligonucleotides used in this study

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted ( left lane ) and depleted ( right lane ) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A 24 -Cru. FMDV A 24 -Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3D pol . c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A 24 -Cru RNA. FMDV A 24 -Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Western Blot, SDS Page

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV 3C pro and 3D pol . a Co-immunoprecipitation of FMDV 3D pol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3D pol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3D pol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3D pol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3D pol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3D pol , starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3D pol and Sam68. (i) Electrostatic surface representation of FMDV 3D pol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3D pol . (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3D pol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3D pol . FMDV 3D pol green docked on Sam68 blue in cartoon representation. The 3D pol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3D pol ( d ) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3C pro , and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Immunoprecipitation, Infection, Western Blot, Control, Sequencing, Negative Control, Binding Assay