EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05. (B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1. (C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced. (D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.
Figure 5A) Representative electron micrographs of entire boutons in wild type, hiw;wnd, and hiw;wnd, Elav-GAL4 > dNmnat RNAi NMJs. B) High magnification electron micrographs showing representative "T-bar" structures at active zones in NMJs of the indicated genotypes. Red arrows denote the beginning and end of the T-bar top, demonstrating changes in T-bar width in the genotypes tested. As illustrated, a significant reduction in T-bar size is observed in hiw;wnd, and this defect is suppressed in hiw;wnd, Elav-GAL4 > dNmnat RNAi. C) Quantification of mean T-bar width [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=13.829, p < 0.0001. WT vs hiw;wnd p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.9318 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)]. (D) Analysis of cumulative frequency distribution of T-bar width shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.5636, p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1815, p = 0.1240 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.4121, p < 0.0001 (****)]. ]. (E) Quantification of mean T-bar height [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=10.472, p < 0.0001. WT vs hiw;wnd p = 0.0008 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.6694 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)] (F) Analysis of cumulative frequency distribution of T-bar height shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.3343, p = 0.001 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1012, p = 0.7748 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.3484, p = 0.0008 (***)].
a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax.figf6(a) ELISA of anti-pneumovax IgM titers in diluted serum 1 day before and 4, 10 and 14 d after intraperitoneal immunization of Atg5f/f and Atg5f/fCD19-Cre mice with 1 μg pneumovax. (b) ELISA of anti-pneumovax IgG3 titers in serum 14 d after injection as in a. Results are presented as absorbance (A). *P 0.05, **P 0.01 and ***P 0.001 (Student's t-test).
Figure 3BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments. cGAS promotes micronuclei generation in BMDMos independently of STING. Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm. cGAS promotes micronuclei generation in BMDMos independently of STING. Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells. γ-irradiation-induced cell death in WT, cGAS-/- BMDMos γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).
HDAC6 KO mouse and knockdown fly developed spontaneous neurodegeneration and protein aggregate accumulation.Figure 7(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm (B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm. (C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI. (D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).
LC3B mediates phagophore elongation whereas GATE‐16 acts downstream.Figure 7(A) Control HeLa cells and HeLa cells stably expressing GFP‐LC3B or GFP‐GATE‐16 were transfected with HsAtg4AC77A‐Myc‐His6. After 48 h interval, the cells were incubated for 2 h in EBSS, and subjected to immunostaining with anti‐Atg16 and anti‐myc followed by confocal microscopy analysis. Quantification of cells containing Atg16 structures larger than 2 μm from three independent experiments is presented at the right panel. Scale bar: 20 μm. (B) HeLa cells were transfected with HsAtg4AC77A‐Myc‐His6 or empty vector (mock). After a 48 h interval, the cells were incubated for 2 h in EBSS, fixed, and their ultrathin sections were analysed by TEM as described in 'Materials and methods'. *P0.05.
a,b) Induction of Atg5 deletion in pneumocytes isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice.f5(a) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot. β-actin is shown as a loading control. (b) Atg5 mRNA expression was determined by quantitative PCR. Data are shown as mean relative mRNA expression±s.e.m. in KRas;Atg5fl/fl tumours as compared with tumours from KRas;Atg5fl/+ mice (set to 1). Values were normalized to β-actin mRNA expression. n=3 per group. (c) GO terms showed enrichment of genes annotated for locomotion and leukocyte migration, cell adhesion, blood vessel remodelling, immune responses, oxidative stress or hypoxia in KRas;Atg5fl/fl as compared with control KRas;Atg5fl/+ pneumocytes. Enrichment and vizualization analysis was performed using GOrilla and REViGO, with colour depth indicating the enrichment significance (P-value). Differentially expressed genes between KRas;Atg5fl/fl and KRas;Atg5fl/+ pneumocytes were selected using DESeq ('blind' variance estimation, fold-change>2, adjP0.05, false discovery rate adjusted for multiple testing).
Figure 2 A) Larval brains at 16 h ALH, in which kin-lacZ was expressed under the control of insc-Gal4, tub-Gal80ts, were labelled with β-Gal, Msps, and Dpn. The primary protrusion of qNSCs was marked by Msps. Quiescent NSCs at the ventral nerve cord (VNC) are shown. Arrows indicate the localization of Kin-lacZ in qNSCs. B) Larval brains at 6 h ALH from insc-Gal4; UAS-nod-lacZ were labeled with β-Gal, Msps, and Dpn. Quiescent NSCs at both CB and VNC are shown. Arrows indicate the localization of Nod-lacZ in qNSCs  C) Representative kymographs decorated with and without coloured lines depicting movements of EB1-GFP comets in the primary protrusion of qNSCs at 0 h ALH  D) Representative kymographs decorated with and without coloured lines tracking movements of EB1-GFP comets in the primary protrusion of qNSCs at 6 h ALH  E) Quantification graph of the percentage of anterograde and retrograde movements of EB1-GFP comets in the primary protrusion of qNSCs at 0 h ALH and 6 h ALH. n=28 at 0 h ALH; n=64 at 6 h ALH. 3 biological replicates per time point. p=0.9096 (ns)  F) Quantification graph of the velocity of EB1-GFP comet movement in the protrusion of qNSCs at 0 h ALH, 6 h ALH and 10 h ALH, as well as in interphase NSCs (aNSCs) at 72 h ALH. n=250 for 0 h qNSCs (anterograde); n=30 for 0 h qNSCs (retrograde); n=211 for 6 h qNSCs (anterograde); n=24 for 6 h qNSCs (retrograde); n=171 for 10 h qNSCs (anterograde); n=103 for interphase NSCs (anterograde). ****p<0.0001; * p=0.0285
Figure 2A In situ hybridization analysis of MdCAX3 in Malus domestica root and stem cross sections. Se, sieve tube; cc, companion cell. Scale bars: 100 μm. B Subcellular localization of MdCAX3 in N. benthamiana leaves. The eGFP-MdCAX3 vector were transformed into A. tumefaciens GV3101 and injected into N. benthamiana leaves. To clarify MdCAX3 subcellular localization, the leaves were stained with DAPI for 5 minutes, and then treated with hyperosmotic (2 M NaCl) for 15 minutes before confocal imaging. N, Nucleus; V, Vacuole. Scale bars: 25 μm.
TAG .Figure 4(A) Inhibition of cholesterol mobilization from the plasma membrane by the ACAT inhibitor SZ58-035. (B) 25HC activates ACAT and stimulates the formation of lipid droplets. Calu-3 cells were cultured in medium containing 2% LPDS prior to the treatment of ethanol or 5 µM 25HC for 16h. Subsequently, cells were stained by LipidSpot to visualize intracellular lipid droplets. Scale bar, 50 μm. (C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting. (D, E) Inhibition of ACAT activity by SZ-58-035 rescues viral entry viral entry. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16 hours in the presence of DMSO or 40 µM SZ-58-035. Then cells were washed and incubated with DMSO or 40 µM SZ-58-305 for 1 h prior to infection with SARS-CoV-2 pseudovirus. Luciferase activity was measured at 24-hour post-infection (D). EGFP signal was captured by fluorescence microscope 24 hours post-infection (E). (F) Knockdown of ACAT1/2 rescues SARS-CoV-2 pseudovirus entry in 25HC-treated cells. Calu-3 cells were transduced with lentiviral vectors carrying non-targeting shRNA or shRNAs targeting ACAT1 and ACAT2. 48h post-transduction, cells were treated with ethanol or 5 µM 25HC for 16 hours. Then the cells were challenged with SARS-CoV-2 pseudovirus. 24h post-pseudovirus challenge, viral entry was quantified by luciferase assays.
PLCβ1 and PTPRN2 promote metastatic breast cancer cell migration as targets of miR-335.Figure 7Figure 7: PLCβ1 and PTPRN2 promote metastatic breast cancer cell migration as targets of miR-335.(A) Model of PTPRN2 and PLCβ1-mediated regulation of plasma membrane PI(4,5) P2 levels to promote metastatic migration through CFL1. In poorly metastatic breast cancer cells, levels of PLCβ1 and PTPRN2 are low. In highly metastatic breast cancer cells, miR-335 is silenced and PTPRN2 and PLCβ1 levels increase. PTPRN2 and PLCβ1 reduce plasma membrane PI(4,5) P2, releasing CFL1 from its inactive state in the plasma membrane to the cytoplasm where it increases actin remodeling.
Figure 3A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests). C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification. D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band). E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA. F Control (IgG) and UBXD1co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.
Figure 4(A) WT or LTalpha-/- mice were treated i.v. with PBS-loaded liposomes (PLL) or clodronate-loaded liposomes (CLL) 42 h prior to s.c. inoculation of the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was analyzed by FFA. Mean ± SEM. Date are pooled from two experiments, n=8-10. Mann-Whitney test; *P < 0.05, ***P < 0.001, ****P < 0.0001. (B) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=10. Two-way ANOVA with Bonferroni's multiple comparisons test; ***P < 0.001, ****P < 0.0001. (C) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of RRV. Viral genomes in the dLN and serum were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8-9. Mann-Whitney test; ***P < 0.001, ****P < 0.0001.
Figure 2a) Clones were categorized as attached or not attached to hair follicles. Average clone size of attached (circles) and non-attached (squares) clones were plotted over time. T-test, *** p<0.0001, ** p<0.001. (Number of clones: 1 week n = 271, 3 weeks n = 1847, 5 weeks n = 517, 12 weeks n = 706, 24 weeks n = 258). Data are represented as mean ± s.d. b) Photomicrographs represent 2D optical sections from a Z stack acquisition of a whole mounted back skin harbouring multiple clones of different colours at 12 weeks. The yellow and blue clone in the IFE immediately adjacent to a HF are considered as attached to the HF. The orange and pink clones are not attached to the HF. Limits of clones (lines) and cells (dots) within each clone were identified on this optical section. White dashed circles represent the projection of hair follicle openings (scale bar= 50 µm). c) Bar charts showing the relative frequency of clone size between 3 and 24 weeks after recombination. In the P42 group, clones attached to HF were more likely to be bigger than those not attached at 3, 5, 12 and 24 weeks. ǂǂǂǂ Chi square test p<0.0001.The number of clones analyzed at each time-point is indicated on the graph. Of note, whether attached or not clones continued to grow over time: ****Chi square test p<0.0001. All data are represented as mean ± s.e.m. d) Plot showing the probability of a clone to be attached to a hair follicle as a function of its size (dashed line: predicted curve based on theoretical probabilities according to spacing of hair follicles at different time-points; squares: experimental data). Between different time points the distance between hair follicles varies slightly; therefore each time point (3, 5, 12 and 24 weeks) has a distinct theoretical curve. The observed frequency of clone attachment to hair follicles is at every time point above the random prediction.
Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cells.Figure 2Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.
Figure 1A. Co-immunoprecipitation of flag-praja2 and GFP-TBC1D31 from lysates of HEK293 cells. The immunoprecipitation (Ip) was performed using an anti-flag antibody or control IgG. B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis. C. Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis. D. Lysates expressing flag-praja2 were subjected to pull down assay with GST and GST-TBC1D31940-970 polypeptides. E. Co-immunoprecipitation of endogenous TBC1D31/praja2/PKAc complex from cell lysates. F. Staining of HEK293 cells with anti-TBC1D31, anti-γ-tubulin and anti-praja2 antibodies. Nuclei were stained with DRAQ5 (blue). Where indicated, cells were transfected with GFP-TBC1D31. Arrows indicate the pool of praja2 colocalizing with TBC1D31 staining at the centrosome. G. Cells transfected with control siRNA (siCNT) or siRNA targeting TBC1D31 (siTBC1D31) were stained for praja2, anti-γ-tubulin and DRAQ5. H. Schematic picture of TBC1D31/praja2/PKA complex.
Complex-centric detection of 20S proteasome assembly intermediates.Figure 6Protein-level SEC chromatograms of the 22 canonical 26S proteasome subunits. Vertical black lines indicate the apexes of six distinct co-elution signals detected in complex-centric scoring; two of which represent well-known co-occurring variants, the full 26S (i) and the 20S (ii) particle devoid the 19S lid and ATPase (Indicated by structural models, PDB accession 5GJR) and four of which, composed of predominantly 20S α and β subunits, appear at reduced size (222 - 107 kDa, fractions 39, 40, 42 and 46). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6B). Zoom in to chromatograms of 20S components in full and reduced MW range and in the context of chaperones known to be involved in assembly according to the current model of 20S biogenesis (lower panel, assembled after Saeki & Tanaka, 2012 (Saeki & Tanaka, 2012) and PDB accession 5GJR), colored by protein class. Reduced MW species are classified into early and late assembly intermediates (as opposed to artifacts of disassembly) by defined co-elution of early assembly chaperone PSMG3/PSMG4 dimer, late assembly chaperone proteasome maturation protein POMP and constitutive chaperone PSMG1/2 dimer. Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells.
Effect of DDX3 knockdown and RK-33 on cell cycle progression and apoptosis.Figure 7A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD. B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD. D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent. F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.
Figure 3 Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)  RNA immunoprecipitation (RNA-IP). BAT isolated from mice at 10 min after intraperitoneal (i.p.) injection of PBS or CL316243 was used for IP with control (Ctrl) or CPEB2 (CP2) IgGs. RT-qPCR analysis of precipitated substances for level of Ucp1 mRNA with the non-target control, Gapdh RNA, as the reference. Data are mean ± SD (3-4-mo-old male mice, n = 3 per group). **p < 0.01, Student's t test  PAT assay to monitor the poly(A) tail length of Ucp1L RNA in BAT isolated from 4-mo-old WT and KO mice at 10 min after i.p. injection of PBS or CL316243. As a poly(A)- control, the oligo(dT)-annealed total RNAs were treated with RNaseH to remove poly(A) tails before PAT assay  Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1S, -mUcp1L, -mUcp1LΔHex or -mUcp1LmutCPE (the 2 CPEs were mutated to UUUUGGC and UUUGCC) 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged full-length (CP2) or RNA-binding domain (RBD) of CPEB2 into differentiated HIB1B adipocytes. Cells were treated ± CL-316243 (CL) before harvesting for luciferase assay. Data are mean ± SD from 3 independent experiments. ap < 0.05 compared with EGFP, bp < 0.05 compared with RBD, cp < 0.05 compared with CP2, one-way ANOVA  Representative polysome profiles from CPEB2-WT and -KO BAT. RT-qPCR analysis of the polysomal distribution of Ucp1L mRNA with RNA isolated from each fraction