sam68 protein  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Sam68 is upregulated in the liver of fasting mice, HFD-induced obese and diabetic mice, and db/db diabetic mice. ( A , B ) Sam68 mRNA (qRT-PCR) ( A ) and protein (( B ), left panel, representative Western blot; right panel, quantification) expression were evaluated in the liver of WT mice under feeding condition or after fasting for 16 h. n = 3. ( C , D ) Sam68 protein expression was evaluated in the livers of ( C ) WT mice on normal diet (ND) or HFD for 12 weeks and of ( D ) db/m (control) and db/db mice at age of 8–12 weeks (left panel, representative Western blot; right panel, quantification). n = 4–5. Data are expressed as mean ± s.e.m. * p < 0.05, *** p < 0.001 (unpaired t test).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Deletion of hepatic Sam68 attenuates glucagon-induced gluconeogenic gene expression. ( A ) Sam68 protein expression in the liver of Sam68 LKO and Sam68 f/f mice (left panel, representative Western blot; right panel, quantification). n = 5–6. ( B ) Blood glucose levels in the mice under feeding condition or after 16-h fasting. n = 8–9. ( C ) mRNA expression (qRT-PCR) of PGC-1α, PEPCK, and G6Pase in the liver of mice after 16-h fasting. n = 4. ( D ) Protein expression of PGC-1α, PEPCK, and G6Pase in the liver of mice under feeding condition or after 16 h fasting (left panel, representative Western blot; right panel, quantification). n = 3. ( E – G ) HepG2 cells were infected with lentiviral vector coding for Sam68-shRNA (KD) or non-targeting shRNA (NT), then the transduced cells were selected in puromycin for 14 days. ( E ) Sam68 protein expression was evaluated (left panel, representative Western blot; right panel, quantification). n = 3. ( F ) Glucose production was measured in the culture media of cells that had been treated with glucagon (100 nM) or PBS for 4 h. n = 3. ( G ) mRNA expression of gluconeogenic genes was measured in cells after treatment with glucagon (100 nM) for 0–3 h ( n = 3). Data are expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant (( B , C , F ), and right panels of ( A , E ): unpaired t test; G and right panel of ( D ): two-way ANOVA).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Gene Expression, Expressing, Western Blot, Quantitative RT-PCR, Infection, Plasmid Preparation, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Sam68 interacts with CRTC2 in the nucleus, enhances CREB/CRTC2 transactivity, and co-occupies on the CRE motif in the promoters of gluconeogenic genes. ( A ) WT and Sam68 –/– primary hepatocytes were treated with glucagon (100 nM) for 30 min, then their nuclear and cytoplasmic fractions of proteins were isolated; Sam68 protein in each fraction was co-immunoprecipitated, and CRTC2 protein in the precipitates was detected by immunoblotting. ( B ) Plasmids coding for CRE-containing promoter-driven firefly luciferase expression and CMV promoter-driven renilla expression were co-transfected with different combinations of pcDNA3-HA (Empty), pcDNA3-HA-Sam68, pcDNA3-Flag-CRTC2, and/or pCF-Flag-CREB plasmids in HepG2 cells; 48 h later, luciferase activity was measured and normalized to renilla activity. n = 3. ( C ) WT and Sam68 –/– hepatocytes were treated with glucagon (100 nM), forskolin (10 μM), or Bt2-cAMP (100 μM) for 30 min; then, Sam68 occupancy on the CRE motif of promoters for PGC-1α, PEPCK, and G6Pase were evaluated via ChIP assay. n = 3. Data are expressed as mean ± s.e.m. ** p < 0.01, *** p < 0.001, ns, not significant (( B , C ): two-way ANOVA).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Isolation, Immunoprecipitation, Western Blot, Luciferase, Expressing, Transfection, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Sam68 promotes gluconeogenesis through CRTC2. pcDNA3-HA-Sam68 or pcDNA3-HA (Empty) plasmid was co-transfected with CRTC2 siRNA or non-targeting siRNA in HepG2 cells; 48 h later, the efficiencies of transfections were evaluated by qRT-PCR for Sam68 ( A ) and CRTC2 ( B ) mRNA expression and by Western blotting for ( C ) Sam68 and CRTC2 protein levels. n = 3. The transduced cells were also treated with glucagon (100 nM), then ( D ) glucose produced in the cell-culture media was measured after 4 h of treatment ( n = 3), and ( E ) mRNA expression of PGC-1α, PEPCK, and G6Pase were evaluated at 0 [basal], 1, 2, and 3 h of treatment ( n = 3 per time point). Data are expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant (two-way ANOVA).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Plasmid Preparation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Produced, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Sam68 shuttling between the cytoplasm and nucleus is regulated by the opposing actions of insulin and glucagon. ( A , B ) WT primary hepatocytes were treated with ( A ) insulin (100 nM) or ( B ) glucagon (100 nM) for 30 min; PBS was used as control treatment. Then, whole cellular, nuclear, and cytoplasmic fractions of proteins were isolated, and the abundance of Sam68 protein in each fraction was analyzed by Western blotting. ( C , D ) WT mice were intraperitoneally injected with ( C ) insulin (1 U/kg body weight) or ( D ) glucagon (30 µg/kg body weight); PBS was used as control injection. Nuclear and cytoplasmic fractions were isolated from liver tissues 20 min after insulin injection or 10 min after glucagon injection, and Sam68 protein expression in each fraction was evaluated (left panel, representative Western blot; right panel, quantification). n = 3. * p < 0.05, *** p < 0.001, ns, not significant (right panel of ( C , D ): unpaired t test).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Control, Isolation, Western Blot, Injection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Deletion of hepatic Sam68 improves systemic insulin sensitivity in HFD-fed mice. Four-week-old Sam68 LKO and Sam68 f/f mice were fed with HFD for twelve weeks and analyzed. ( A , B ) Blood glucose levels were measured in mice ( A ) under feeding conditions or after 16 h fasting ( n = 9–10) and ( B ) in the ITT (insulin tolerance test; n = 7–8). ( C – E ) Sam68 LKO and Sam68 f/f mice were intraperitoneally injected with insulin (+) or PBS (–); 20 min later, ( C ) liver ( n = 3–5); ( D ) epiWAT ( n = 3–4), and ( E ) skeletal muscle tissues ( n = 3–4) were harvested, and the protein levels of phosphorylated AKT (at amino acids Ser-473 and Thr-308) and total AKT were evaluated (left panel, representative Western blot; right panel, quantification). Data are expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant (( A ): unpaired t test; ( B – E ): two-way ANOVA).

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Injection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity

doi: 10.3390/ijms231911469

Figure Lengend Snippet: Schematic presentation of molecular mechanisms underlying Sam68 subcellular shuttling and Sam68-mediated hepatic gluconeogenesis. Our results suggest that Sam68 shuttling between the nucleus and cytoplasm is regulated by the opposing actions of glucagon and insulin and plays an important role in the regulation of hepatic gluconeogenesis and glucose production. Specifically, glucagon signaling triggers Sam68 translocation into the nucleus, where Sam68 interacts with CRTC2 to augment CREB/CRTC2 transactivity and gluconeogenic gene expression, promoting glucose production. On the other hand, insulin signaling promotes Sam68 export to the cytoplasm, diminishing CREB/CRTC2 transactivity and gluconeogenesis. In diabetes, hepatic Sam68 expression is elevated, and the skewed metabolic signaling, i.e., increased glucagon signaling and/or decreased insulin signaling, induces Sam68 translocation into the nucleus, where it augments CREB/CRTC2 transactivity, leading to hyperglycemia. GCGR: glucagon receptor; AC: adenylyl cyclase; PKA: protein kinase A; SMEK/PP4C: suppressor of MEK null/protein phosphatase 4 catalytic subunits; PP2B: protein phosphatase 2B.

Article Snippet: Then, 1 mg total protein was incubated with protein A/G plus agarose conjugated Sam68 antibody (Santa Cruz, CA, USA) at 4 °C for overnight and washed 4 times with lysis buffer; the immunoprecipitants were eluted by boiling for 5 min, and extracts were analyzed by Western Blotting.

Techniques: Translocation Assay, Gene Expression, Expressing

Journal: Genome biology

Article Title: Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing.

doi: 10.1186/s13059-020-02199-6

Figure Lengend Snippet: Fig. 6 NimtRNA protein interaction analysis. a Biotinylated transcripts containing or lacking nimtRNAs were incubated with nuclear protein extracts. Subsequently, binding proteins were isolated and analyzed by PAGE; differential band patterns were excised and analyzed by MS. b GO analysis of nimtRNA transcript interacting proteins. c The top 10 nimtRNA transcript interacting proteins ranked by absolute abundance. d–f An electrophoretic mobility shift assay was performed with increasing concentrations of KHDRBS1 being incubated with a transcript of the nimtRNATyr (d), nimtRNATyr delT (e), or snoRNA SNORD115 (f). The unbound transcript is indicated by an asterisk; the transcript-protein complexes are indicated by triangles

Article Snippet: Human recombinant KHDRBS1 (Sam68; CAT#: TP300263) was ordered from Origene (Rockville, Maryland, USA).

Techniques: Incubation, Binding Assay, Isolation, Electrophoretic Mobility Shift 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) 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