EMBO/sd-character-level-ner huggingface dataset:
(F) RNA isolated from kidneys treated with doxorubicin or abemaciclib and mRNA encoding indicated p53-associated SASP genes quantified by qRT-PCR (n=6 mice/group). (G) RNA was isolated from kidneys of p16-3MR mice treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days), and mRNA encoding p53 associated SASP genes was quantified by qRT-PCR and normalized on tubulin (n=5 mice/group). ( Data information: Data are means ±SD. Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.
A FRAP images of CAF-1 bodies to indicate the internal diffusion property. GFP-tagged CHAF1A was overexpressed in HEK293T cells. Twenty-four hours later, live cells were proceeded to time series imaging. One of the CAF-1 bodies was bleached with intense laser pulse. Images of live cell were captured every 4 seconds. One of the unbleached CAF-1 bodies was used as control. Data represented eight time points. At least three cells of each sample were treated with intense laser pulse to obtain bleached CAF-1 bodies.
(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.
(I) Similar experiments as in (C) were performed in W1118 and TBPH-/- adult flies. The mRNA levels of lysosomal and autophagic genes were quantified and normalized relative to GAPDH. The data from three independent experiments are presented as means ± S.E.M., ns, not significantly different; **, p<0.01, one-way ANOVA.Total RNA was prepared from W1118 and TBPH-/- larvae and subjected to qRT-PCR analysis. The level of TBPH and raptor were quantified and normalized relative to RPL32. Data from three independent experiments represented as means ± S.E.M., **, p<0.01, one-way ANOVA.
Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).
(A) Alternative splicing of sams-3, sams-4 and sams-5 in smg-2 (yb979) and smg-2 (yb979); sams-5 (gk147); sams-1 (ok2946) mutants. Synchronized L1 larvae of each strain were incubated in S-complete medium alone (lanes 1 and 5), with OP50 (OD600=10.0) (lanes 2 and 6) and with OP50 supplemented with 25 mM L-Met (lanes 3 and 7) or 25 mM cycloleucine (cLeu) (lanes 4 and 8) for 3 hours at 20°C. The splicing patterns were analyzed and presented as in Figure 2B (n=3).
(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.
B Two or six (-97m3) representative primary transformants expressing GUS under the given PAP8 promoter version; FC+, the corresponding promoters were tested positive in functional complementation of the mutant pap8-1. Scale bar equals 3 mm.
E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.
U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.
E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001
(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.
A Scheme of H1-GFPFRAP in heterochromatin foci (with HP1a-mCherry) and euchromatin (without HP1a-mCherry). The relative FRAP curves of euchromatin (Eu) and heterochromatin (Het) are shown divided into two parts-the mobile fraction (MF) and the immobile fraction (IF) after bleaching recovery. (ROI, region of interest)
(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.
C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).
(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.
One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).
Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.
Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.
B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.
D Cellular localization of Zn2+ (blue) in Mx and MdCAX3-suppressed Mx roots under Fe deficient conditions. Arrowhead indicates the cytoplasm. Scale Bars: 20 μm. E Fluorescence intensity detection at the position indicated by the long and narrow arrow in the Fig 6D.
Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included.
D The S622A mutation strongly reduces phosphorylation by PBL27. Each of phosphorylation sites was substituted by alanine, and subjected to the in vitro phosphorylation assays.
(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.
(C) Viral RNA content as determined in various organs at 3 dpi (n = 6/group) by use of conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR detection limits. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).
A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.
B. Intracellular survival of EGD-(pADc-GFP), EGD-(pADc-inlK), ΔactA-(pADc-GFP) and ΔactA-(pADc-inlK) in RAW 267.4 macrophages. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).
The upper part of the table shows the PWMs over-represented (p-values determined by a two-tailed Welch's t-test are shown in red) in Lo-G PDAC cell lines relative to both enhancers specific of Hi-G PDACs (p vs. Hi-G) and the FANTOM 5 collection of human enhancers (p vs. hs enh) (Andersson et al, 2014). The frequency (f) of these PWMs in the enhancers specific of Lo-G and Hi-G PDACs is also shown. The cognate TFs assigned to these over-represented PWMs are indicated in bold if over-expressed in Lo-G PDACs. The bottom part of the table shows the result of the statistical over-representation analysis for those TFs expressed selectively in Lo-G PDACs but whose PWMs were not over-represented in this analysis (p >1E-5 determined by two-tailed Welch's t-test).
(F) Mean cfu of Pto DC3000 2 days after syringe inoculation and pre-treatment with 1 μM GLV2; n=20 biological replicates from 5 pooled experiments -/+ SD (Students T-test, *p <0.05).
(D) HeLa cells KO for LCS were transfected with indicated LCS RUSH constructs for 16 hours, and expressed enzymes were retained in the ER (-biotin) or placed in the Golgi by addition of biotin (40 μM) (+biotin) and SL output monitored by [3H] - sphingosine pulse-chase assay. The ratio of GM/Gb is represented.
F Endothelin-1 was increased in lung tissue after siISCU delivery under normoxic conditions (N = 5/group), **P = 0.0027.
J Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).
d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001).
(A, B and C) Plasma levels of 3-hydroxybutyrate in WT and p38γ/δ-/- (A), Lyzs-Cre and p38γ/δLyzs-KO mice (B) and Lyzs-Cre Ly6G Ab treated after MCD diet (C).
(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).
J A model for the function of nSBs in splicing control. Thermal stress-induced HSATIII integrates subsets of de-phosphorylated SRSFs and SR-related proteins. After stress removal, CLK1 relocates to nSBs to promote rapid re-phosphorylation of the sequestrated SRSFs, especially SRSF9. Re-phosphorylated SRSF9 suppresses splicing of target introns to accumulate intron-retaining RNAs in the nucleus.
Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.
A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.
(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.
(C) ATP assay for dystrophic muscle cells in the presence of MOTS-c (n=6; *p<0.05). NC refers to untreated H2K or H2K mdx cells.
All diagrams are aligned at the left and at the same scale, using the full-length E protein (Ev, top line) and sE401v (second line), derived from virions, as reference. Recombinant constructs are shown below the dashed line: sE400r, sE401r, sE404r (containing 6 N-terminal amino acids of H1), sE412r (containing H1), sE419r (containing H1 and H2), sE428r (containing H1, H2 and CS) and sE448r (containing the whole stem). Abbreviations EK and StT indicate heterologous sequences corresponding to an enterokinase-specific cleavage site and a strep-tag used for affinity purification, respectively, added at the C-terminal end of each construct.
B. UMAP (Left) and RNA velocity (Right) of chorion cells where 5 unsupervised clusters were identified.
C) Imaging of IBIS1‐mCherry‐expressing P. berghei reveals similar features of the LS‐TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26 h post‐infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10 µm.
B. Distribution of Translation Efficiency Score (TES) across novel lncRNAs identified in this study and RefSeq genes. Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend till 5th and 95th percentiles.
(B) Schematic diagram of the in vitro fusion assay of ATG16L1 and mATG9 vesicles. Postnuclear supernatants (PNS) of cells expressing mStrawberry-ATG16L1 were mixed with PNS from cells expressing ATG9L1-GFP for 1 hr in the presence of ATP, ATP and NEM at 37°C, or with ATP on ice. The samples were fixed and mounted on a microscope slide with Mowiol 4-88 for confocal analysis.
(C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test.
(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.
(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.
C. Schematics of Upf1 fragments used for the in vitro interaction assay; yUpf1-HD is the helicase domain (220-851) of the yeast Upf1 protein, hUpf1-HD is the helicase domain (295-914) of human Upf1 (not to scale)
Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.
SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNA. Scale bar, 100 μm.Quantification of the numbers of senescent cells shown in (C). Error bars represent SEM (n = 3). **P 0.01; ***P 0.001.Quantification of the numbers of spheres formed by cells shown in (C). Error bars represent SEM (n = 3). *P 0.05; **P 0.01; and ***P 0.001. Statistical significance was assessed by Student's unpaired t‐test.
The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF.
The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis. Semi-thin sections of PLP/CFA and CFA-injected mice were probed with the indicated antibodies (A, B; see also Appendix Fig. S13)
Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618)
A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.
(B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32.
Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).
(B) Longer HTT polypeptides are modified by SUMO-1. Western blot overexpressing HTTex1p (46QP-H4 or 3R), unexpanded HTT-586 fragment (25Q-586 or 25Q-3R-586 aa), and expanded HTT-586 fragment (137Q-586 or 137Q-3R-586 aa) with SUMO-1 (GFP-SUMO-1). Cell lysates were subjected to HTT immunoprecipitation (IP) using HTT antibody. Western analysis performed with the Odyssey (LI-COR) allows detection of HTT (data not shown) and SUMO simultaneously and shows that all forms of HTT are SUMO-1 modified using the anti-GFP antibody. HTTex1p is covalently SUMO-1 modified (lane 3), and the modification disappears when Lys are mutated to Arg (3R) (lane 4). Both unexpanded (lane 4 and 5) and expanded HTT-586 fragments (lane 6 and 7) are covalently SUMO-1 modified in both the presence and absence of the three Lys in the N-terminal region of HTT (3R). Free SUMO-1 is indicated with the arrow, and SUMO-modified HTT is indicated by the boxes. Inset on the right, from a replicate experiment, shows comigration of expanded HTT (anti-HTT) and SUMO-1 (anti-GFP) displayed in the gray scale and in color when the two antibodies are merged (HTT in red, SUMO-1 in green, and yellow when colocalizing).
GFP-LC3 expressing mice were i.p. injected with 3,4-DC for 24 h. Leupeptin (Leu) was used to test autophagic flux in vivo and GFP-LC3 dots were measured in liver tissue. Data are means ± SEM of at least three mice (*= p < 0.05 versus ctr without Leu; #= p < 0.05 versus ctr with Leu).
Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody.
Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).
A. Strategy for identification SPATA2-dependent protein association with the TNF-RSC. SPATA2 was knocked down using RNAi, cells were stimulated with FLAG-TNF-α, and the TNF-RSC was enriched from control and SPATA2 knockdown cells as in Figure 3A. The enriched proteins were quantified using SILAC-based MS.
Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Unpaired t-test, ns = not significant. Data are means ± SEM.
(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.
Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).
(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).
(A) Cartoon depicting the principle of the endosomal PI4P BRET measurement. The Venus fluorescent protein is targeted to the respective endosomes using Rab proteins, while Renilla luciferase [termed Super luciferase (Sluc)] is fused to the P4M2x PI4P reporter. This reporter binds to PI4P the majority of which is in the PM. The PI4KA inhibitor, GSK-A1 (A1) reduces PI4P in the PM allowing more of the reporter to find the endosomal PI4P that is produced by PI4Ks that are insensitive to GSK-A1. Relocation of the P4M2x reporter tagged with Sluc to the PI4P-rich endosomes will increase the energy transfer between the enzyme and Venus in the presence of the coelenterazine H substrate.
(F) Western blot analysis of UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (D).