Journal: bioRxiv

Article Title: CBP/EP300 acetylates and stabilizes the stress-responsive Heat Shock Factor 2, a process compromised in Rubinstein-Taybi syndrome

doi: 10.1101/481457

Figure Lengend Snippet: (A) The ectopically expressed YFP-HSF2 protein is acetylated by exogenous HA-CBP or EP300. Representative immunoblots (n=5 independent experiments). HEK 293 cells were transfected with different combinations of YFP-HSF2, HA-CBP, HA-EP300 constructs, and mock-HA or -GFP constructs. YFP-HSF2 was immunoprecipitated using anti-GFP-trap antibody (IP GFP-Trap) and its acetylation status was determined by WB analyses, using an anti-pan-acetyl-lysine antibody (AcK; left panels). Immunoprecipitated HSF2 was detected using an anti-GFP antibody (WB: GFP). The total amounts of the proteins in the input samples were detected with anti-GFP or anti-HA antibodies (inputs; right panels). Actin, loading control. C TA :Trap®-A beads were used as a negative control (see Experimental Procedures). (B) The HSF2-Myc protein is not acetylated by a dominant-negative form of CBP (DNCBP-HA). HEK 293 cells were transfected as in ( A ), except that HSF2-Myc was used instead of HSF2-YFP, and immunoprecipitation was performed using anti-Myc-trap antibody (IP Myc-Trap). Representative immunoblots (n=2 experiments). C TA : Trap®-A beads were used as a negative control (see Experimental Procedures). HSP90: loading control. (C) Schematic representation of the eight main acetylated lysine residues of the HSF2 protein. Purified mouse Flag-HSF2, co-expressed with HA-EP300, immunoprecipitated and subjected to MS analysis for detection of acetylated lysine residues. The three lysine residues K128, K135, K197, located in the oligomerization domain (HR-A/B), are enlightened in red and K82, located in the DBD, in blue; the other four lysine residues (K209/K210, K395/K401) are indicated in regular black. The DNA-binding domain (DBD, orange); the oligomerization domain (HR-A/B; green) and the domain controlling oligomerization (the leucine-zipper-containing HR-C; green); as well as the N-terminal domain (activation domain TAD; red) are illustrated. The boundaries of each domain are indicated in bold and blue. Bold and blue numbers correspond to the number of the amino acids located at boundaries of the domains of the mouse HSF2 protein, numbered from the +1 (ATG); the equivalent in the human HSF2 protein, if different, are indicated in bold and black. These four (K82, K128, K135, K197) or three lysine residues (K128, K135, K197) were mutated into glutamines (4KQ or 3KQ, respectively) or arginines (4KR or 3KR, respectively; see also Figure S2A). (D) The mutations of three or four lysine (K) residues to arginine (3KR or 4KR) or glutamine residues (3KQ or 4KQ) decrease global HSF2 acetylation levels. HEK 293 cells were co-transfected with EP300-HA and wild-type (WT) or mutated human HSF2-Myc on the indicated lysine residues. After immunoprecipitation of HSF2, using anti-Myc antibody, its acetylation was analysed by WB using an anti-AcK antibody. HSC70, loading control. n=3 independent experiments. (See also Figure S2B,C). (E) In vitro acetylation of HSF2 peptides containing the K135 or K197 residues by recombinant CBP Full-HAT . Time course of reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of the acetylation of HSF2K197 (upper panel) and HSF2K135 peptides (lower panel) by CBP Full-HAT. Aliquots of the reaction were collected at 0 (black), 1 (red) or 2 (green) hours and elution of peptides was monitored by fluorescence emission at 530 nm (excitation: 485 nm, uV: arbitrary unit of fluorescence; see Figure S2D for HSF2K82 peptide). (F) Quantification of the in vitro acetylated HSF2 peptides containing K82, K135, and K197 residues. The AUC (area under the curve) of the acetylated K82, K135 and K197 peptides was quantified and converted in product concentration using a calibrated curve of various known concentrations of peptides. Note that it was not possible to investigate the acetylation of the HSF2 K128 peptide by CBP, because this peptide was repeatedly insoluble at the synthesis steps (Manufacturer’s information; see also Figure S2D-F).

Article Snippet: Cells were lysed in Lysis buffer (50 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5 %, Glycerol 10 %, VPA (1 mM), DTT 1 mM, PMSF 1 mM, proteases inhibitors, phosphatase inhibitors (Roche)) and then, HSF2 was immunoprecipitated using anti-GFP- or anti-Myc-trap antibody, or as a control Trap®-A control (ChromoTek).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Negative Control, Dominant Negative Mutation, Purification, Binding Assay, Activation Assay, In Vitro, Recombinant, Liquid Chromatography, Fluorescence, Concentration Assay

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: UbiScan for global identification of LIN41 ubiquitination substrates. ( a ) Proteomic strategy for global identification of LIN41-mediated ubiquitination targets under self-renewal conditions (LIF) and during neural differentiation with RA using a mouse ES cell line with Dox-inducible expression of LIN41-Flag. Each extract was processed by digestion with trypsin followed by affinity purification using an antibody specific for lysine residues carrying a diglycine linkage (K-GG). Enriched ubiquitin-modified peptides were analyzed by spectrometry. ( b ) iLin41 protein extracts prepared for the UbiScan were immunoblotted with α -LIN41 and α -OCT-4 antibodies to validate LIN41 induction by Dox and the conversion of pluripotent ES cells to differentiated cells after 3 days of RA treatment, respectively. ( c ) Venn diagram compares the sets of proteins showing a ≥2.5-fold increase in ubiquitination after LIN41 induction between the pluripotent and differentiated cell states. ( d and e ) Functional annotation analysis depicting significantly enriched Panther pathways among proteins with ≥2.5-fold more ubiquitination upon LIN41 induction in pluripotent ( d ) and differentiated ( e ) cells. Similar results were obtained for enrichment of p53-related KEGG or REACTOME pathways using Gene Set Enrichment Analysis ( http://software.broadinstitute.org/gsea/msigdb/index.jsp , data not shown). ( f ) Diagram summarizing selected p53 pathways with LIN41 ubiquitination candidates from UbiScan depicted in blue and their functional relationships (arrow=direct activation, dotted arrow=indirect activation, inhibition line=direct negative regulation). ( g ) Fold change ratio of Dox + versus Dox − in differentiated cells for the indicated diglycine-modified sites within p53 and schematic of p53 domain structure. DBD, DNA Binding Domain; NLS, Nuclear Localization Sequence; TAD, Transactivation Domain; TET, Tetramerization domain. See also and

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Ubiquitin Proteomics, Expressing, Affinity Purification, Modification, Functional Assay, Software, Activation Assay, Inhibition, Binding Assay, Sequencing

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: LIN41 negatively regulates p53 protein levels by ubiquitination ( a ) Induction of LIN41 in differentiated, but not in pluripotent, ES cells downregulates endogenous p53 protein levels (compare lane 5 with 6 and lane 7 with 8) in a proteasome-dependent manner (compare lanes 3 and 4 with lanes 7 and 8). Expression levels of p53, LIN41, OCT-4 and GAPDH were determined by immunoblotting. A short and long exposure are shown for the p53 blot. ( b and c ) Quantification of unmodified ( b ) or ubiquitin-modified ( c ) p53 from LIN41-induced (Dox + ) relative to untreated cell extracts. Relative band intensity was quantified from immunoblots, cell treatments as in a . Data from three independent experiments are presented as means±S.D. values. * P <0.05, ** P <0.01, *** P <0.001. ( d ) LIN41 induction does not affect p53 transcript levels in either pluripotent or differentiated ES cells. p53 mRNA was quantified by qRT-PCR using Oaz1 mRNA for normalization. Data from three independent experiments are presented as means±S.D. values. ( e ) p53 is reduced in response to LIN41 induction during SFEBq cortical organoid differentiation. iLin41 organoid cultures were differentiated with and without Dox. p53 and LIN41 protein levels were monitored at indicated time points by immunoblotting. ( f ) LIN41 induction during neural differentiation promotes p53 poly-ubiquitination in vivo . Multi- and poly-ubiquitinated proteins from pluripotent and differentiated iLin41 cells were enriched using TUBEs after p53 induction with 1 μ M doxorubicin and 4 h MG132 treatment. Ubiquitinated p53, auto-ubiquitinated LIN41 and pull-down efficiency were monitored by immunoblotting. ( g ) Mono- and poly-ubiquitinated proteins were purified using α -Ub-FK2-agarose beads capable of detecting all linkage types. iLin41 cells were prepared as for TUBE assay but without p53 induction. Western blots of input lysates and eluates were probed with a goat α -p53 antibody. Immunoblots of LIN41 and OCT-4 were included as positive and negative control, respectively. LIN41 induction increases ubiquitination levels of endogenous p53

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Modification, Quantitative RT-PCR, In Vivo, Purification, Negative Control

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: LIN41 co-localizes and physically interacts with p53. ( a ) iLin41 cells were immunostained for endogenous p53, LIN41 and nuclear marker Draq5. ( b ) Flag-tagged LIN41 co-precipitates endogenous p53 in HEK293 cells. Co-precipitation of endogenous proteins after IP with either control IgG or α -Flag agarose was demonstrated by western blotting. ( c ) Flag-tagged p53 co-purifies endogenous LIN41. P19 cells were transfected with Flag-p53 or positive control Flag-Mov10. After α -Flag-IP, LIN41 co-purified with similar efficiency using either MOV10 or p53 as bait. ( d ) p53 from mouse ES cells was affinity purified using a mouse-specific p53 Trap. A human-specific p53 Trap served as negative control. Endogenous interaction was observed between p53 and LIN41, but not with GAPDH. ( e ) Schematic representation of LIN41 domains and the deletion constructs used to map the p53 interaction. ( f ) p53 interacts with the NHL domain of LIN41. LIN41-GFP fusion proteins containing the full-length protein or deletion constructs without the NHL, the RING motif or with only the NHL domain were co-expressed with Flag-tagged p53 in HeLa cells. GFP was included as a negative control. Input and bound proteins after Flag-IP were analyzed by western blotting with the indicated antibodies. ( g ) p53 directly interacts with LIN41. GST pull-down assays of in vitro synthesized LIN41 using GST only, p53-GST fusion protein or MDM2-GST. The presence of LIN41 in bound and unbound fractions was analyzed by immunoblotting. See also

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Marker, Control, Western Blot, Transfection, Positive Control, Purification, Affinity Purification, Negative Control, Construct, In Vitro, Synthesized

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: LIN41 affects proliferation and p53-dependent apoptosis during ES cell differentiation. ( a and b ) LIN41 differentially promotes proliferation in differentiating compared to pluripotent cell states. Dox-inducible iLin41 cells were cultured under proliferative ( a ) or differentiation conditions ( b ). Cell number was determined at 24 h intervals in triplicates. Data are presented as means±S.D. of three independent experiments. * P <0.05. ( c ) CASP3 activation during neural differentiation is p53 dependent. p53 WT and p53-deficient mES cells were cultured under proliferative or differentiation conditions. Cleaved CASP3 levels were determined by immunoblotting. ( d ) Induction of LIN41 during neural differentiation reduces CASP3 activation. Cleaved CASP3, p53, LIN41 and GAPDH levels of pluripotent and differentiated iLin41 cells without or with Dox treatment were compared by immunoblotting. ( e ) LIN41 inhibits p53-mediated apoptosis in response to stress. iLin41 cells differentiated in the presence and absence of Dox were pulse treated with 1 μ M doxorubicin. p53 levels and CASP3 activation were determined by immunoblotting. ( f ) LIN41 induction in RA-differentiated stem cells reduces levels of p53 target mRNAs. Pluripotent and differentiated populations of iLin41 cells were cultured without or with Dox. mRNA levels of direct p53 transcriptional targets ( Puma and Noxa ) or the non-transcriptional target ( Bim ) within the apoptosis pathway were analyzed by qRT-PCR and normalized to Oaz1 . Data are presented as mean values±S.D. from three independent experiments. * P <0.05, *** P <0.001. ( g ) LIN41 counteracts p53's transactivation capacity in response to stress. iLin41 cells differentiated for 3 days in the absence or presence of Dox were pulse treated with 1 μ M doxorubicin. mRNA levels of Puma, Noxa and Bim were measured by qRT-PCR assays and normalized to Oaz1 . Data are presented as mean values±S.D. from at least three independent experiments. ** P <0.01, *** P <0.001. See also

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Cell Differentiation, Cell Culture, Activation Assay, Western Blot, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: Loss of LIN41 elevates p53 steady-state levels and reduces p53 ubiquitination. ( a ) Comparison of p53 expression in undifferentiated wild type, heterozygote and homozygote Lin41 -mutant ES cells shows a gene dose-dependent increase of p53 protein levels in Lin41- deficient cells. ( b ) Lin41 deficiency in mouse ES cells does not affect p53 transcript levels analyzed by qRT-PCR. Expression levels were normalized to Oaz1 mRNA. Data are presented as means±S.D. values from three independent clonal ES cell lines per genotype. ( c ) Constitutive levels of the p53 effector Noxa are increased in Lin41 -deficient ES cells. Expression levels of Puma, Noxa and Bim were measured by qRT-PCR and normalized to Oaz1 . Data are presented as means±S.D. values from three independent clonal ES cell lines per genotype. ** P <0.01. ( d ) Cre-induced disruption of LIN41 in Lin41 fl/fl ; Rosa26-CreER T2 mES cells upregulates p53 steady-state levels. Conditional Lin41 knockout cell lines were cultured under proliferative and differentiation conditions. For the induction of Cre-mediated recombination, cells were treated with 500 nM 4-hydroxytamoxifen. p53, OCT-4 and LIN41 protein expression were examined in untreated cells (lanes 1 and 5), in two independent cell lines with stable Lin41 deletion (lanes 2 and 6 plus 3 and 7) or acute loss of Lin41 (lanes 4 and 8). ( e ) Quantification of p53 levels in two independent lines with stable Cre-induced Lin41 deletion relative to untreated cells. Relative band intensity was quantified from immunoblots from three independent experiments. Data are presented as means±S.D. values. * P <0.05, *** P <0.001. ( f and g ) Loss of LIN41 reduces p53 ubiquitination in vivo . After 4 h MG132 treatment, total ubiquitinated proteins from 2-day RA-treated Lin41 fl/fl ; Rosa26-CreER T2 cells were enriched using α -Ub-FK2-agarose beads or TUBEs, as indicated. Ubiquitination levels of endogenous p53, LIN41 and pull-down efficiency were detected by immunoblotting

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Ubiquitin Proteomics, Comparison, Expressing, Mutagenesis, Quantitative RT-PCR, Disruption, Knock-Out, Cell Culture, Western Blot, In Vivo

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: Increased levels of p53 during brain development in the absence of Lin41. ( a ) Lin41 is essential for cranial neural tube closure. Failure of neural tube closure (asterisk) and growth retardation in Lin41 −/− embryos in comparison to control Lin41 +/+ littermate at E9.5 gestation. ( b ) Upregulated p53 expression during neural tube closure of Lin41 -deficient embryos. Transverse cranial sections of E10.5 heterozygote and homozygote knockout Lin41 GT;lacZ transgenic embryos were processed for immunohistochemistry, results for p53 and DAPI are shown. Lin41 mutants display neural tube closure defects (asterisk). ( c ) Higher magnification of b to better visualize p53 and LIN41 expression in the neuroepithelium. Lin41 -deficient embryos exhibit increased nuclear levels of p53. In merged views, p53 is shown in green, β -galactosidase ( β -Gal) from the Lin41 -gene trap allele in red and DAPI in blue

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Comparison, Control, Expressing, Knock-Out, Transgenic Assay, Immunohistochemistry

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: LIN41 affects embryonic apoptosis and the expression of the pro-differentiation gene Grhl3 . ( a ) Widespread apoptosis during neural tube closure of Lin41 -deficient embryos. Transverse cranial sections of E10.5 heterozygote and homozygote knockout Lin41 GT;lacZ transgenic embryos were processed for immunohistochemistry, results for activated CASP3 and DAPI are shown. Lin41 mutants display accumulation of CASP3-positive cell clusters. ( b ) Higher magnification of a to better visualize apoptosis in the optic vesicle ( i ) and neuroepithelium (ii–iii) of Lin41 -deficient embryos. In merged views, activated CASP3 is shown in red, β -galactosidase ( β -Gal) from the Lin41 -gene trap allele in green and DAPI in blue. ( c ) Grhl3 , but not Grhl2 , is responsive to p53 induction in ES cells undergoing neural differentiation. qRT-PCR analysis of Grhl2 and Grhl3 mRNAs following doxorubicin treatment. Lin41 and p53 transcript levels are unaffected, as expected. ( d ) LIN41 induction in RA-differentiated stem cells reduces levels of Grhl 3 mRNAs. Pluripotent and differentiated populations of iLin41 cells were cultured without or with Dox. mRNA levels of Grhl3 were analyzed by qRT-PCR and normalized to Oaz1 . ( e ) LIN41 loss of function in mouse E9.5 embryos leads to elevated Grhl3 mRNA levels. Grhl3 , Lin41 and p53 transcript levels were analyzed by qRT-PCR and normalized to Oaz1 . All data are presented as mean values±S.D. from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001. See also

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Expressing, Knock-Out, Transgenic Assay, Immunohistochemistry, Quantitative RT-PCR, Cell Culture

Journal: Cell Death and Differentiation

Article Title: The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

doi: 10.1038/cdd.2017.54

Figure Lengend Snippet: Working model for the interplay of LIN41 and p53 during neural commitment and neural tube closure. Physical interaction between LIN41 and p53 leads to ubiquitination and inactivation of p53 in cells undergoing neural differentiation. By targeting p53, LIN41 antagonizes pro-apoptotic (CASP3) and pro-differentiation effectors ( Grhl3 ), two pathways downstream of p53 with essential roles in neural tube closure. These results indicate that LIN41 is vital to dampen p53 responses and limit the extent of apoptosis in both ES cell differentiation and mouse embryogenesis, which may account for the embryonic lethality and NTDs of Lin41 - Null mice

Article Snippet: Subsequently, 25 μ l of equilibrated p53 Trap_A (Chromotek, Martinsried, Germany) bead slurry was added per reaction and incubated for 1 h at 4 °C. p53 C-term-Trap_A reacts with both human and mouse p53, whereas p53 N-term Trap_A only detects human p53 and therefore served as a control for unspecific binding.

Techniques: Ubiquitin Proteomics, Cell Differentiation

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Predicted secondary structure of the candidate snoRNAs used for snoMEN vectors. The characteristic features of members of the box C/D and box H/ACA snoRNAs are shown. Conserved box C & D and H & ACA motifs are indicated by orange and blue boxes, respectively. The positions of rRNA complementary sequences are indicated by a green bar. The positions of the M box regions that can be altered to modulate target gene expression (snoMEN vector) are indicated by a cyan bar. ( b ) The vector structure used for targeted suppression of GFP fusion proteins. The sequence in wild type U47 snoRNA that is complementary to pre-mRNAs was changed from 5’-AUAUAAUGAUAUCACUGUAAAAC-3’ to 5’-UCACCUUGAUGCCGUUCUUCUGC-3’ (U47 snoMEN). The resulting U47 snoMEN was subcloned into the 5’ region of the vector with mCherry fluorescent protein cDNA. Diagram shows complementary regions of U47 snoMEN targeting the GFP cDNA sequence (Anti-GFP mRNA). A mutant U47 snoMEN expression plasmid with the box D core motif mutatated was also constructed, shown as BoxDmut on the sequence. The motif labels are the same as in ( a ).

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Targeted Gene Expression, Plasmid Preparation, Sequencing, Mutagenesis, Expressing, Construct

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Live cell images of HeLa GFP , HeLa YFP-Fibrillarin (FBL) , and HeLa GFP-Dyskerin (DKC) stable cell lines that stably express free GFP, YFP-Fibrillarin and GFP-Dyskerin, respectively. ( b ) Extracts were prepared from HeLa cell lines stably expressing either YFP-Fibrillarin, or free GFP, for box C/D snoRNAs (U47, U77, HBII-180C) and either GFP-Dyskerin, or free GFP, for box H/ACA snoRNAs (ACA16 and miR566), after transfection of snoMEN expression vectors, as indicated at the top (U47snoMEN, U77snoMEN, ACA16snoMEN, miR-566snoMEN, and HBII-180CsnoMEN). Extracts were immunoprecipitated using a monoclonal anti-GFP antibody conjugated to beads (GFP-TRAP_A), with the specificity confirmed by western blotting [ , ]. Quantitative RT-PCR was used to detect co-precipitated U47 snoMEN, U77 snoMEN, ACA16 snoMEN, miR566 snoMEN and HBII-180C snoMEN snoRNAs (snoMEN), using U3 (box C/D) and E2 (box H/ACA) snoRNAs as a positive and negative control, respectively, for fibrillarin/dyskerin-associated RNAs. Equal amounts of material were loaded on the IP and Input lanes.

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Negative Control

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: Knock-down efficiency of each of the snoMEN plasmids targeted to GFP (U47snoMEN, U77snoMEN, ACA16snoMEN and miR-566snoMEN at the top of each panel) was tested by western blotting. Detection of protein levels for GFP-SMN1 following transfection of HeLa GFP-SMN stable cell lines, using either mCherry and wild type snoRNA expression plasmid (control), snoMEN expression plasmid (U47snoMEN, U77snoMEN, ACA16snoMEN and miR566snoMEN) and mutant snoMEN expression plasmid (U47snoMEN box Dmut, U77snoMEN box Dmut, ACA16snoMEN box Dmut and miR566snoMEN box Dmut). An equivalent amount of HeLa GFP-SMN extract was loaded for each lane and the proteins separated by SDS PAGE, electroblotted onto membrane and probed both with a monoclonal anti-GFP antibody and with anti-tubulin as a loading control. Graph shows average ratio of GFP-SMN1 signal intensity, normalised using the tubulin signal, which calculated from three independent experiments. P -values are significant according to the Student's t -test; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Knockdown, Western Blot, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Control, Mutagenesis, SDS Page, Membrane

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Expression level of endogenous SMN1 and GFP-SMN1 were measured by western blot analysis. Images show an example of a western blot, with shorter exposure in the upper panel and longer exposure in the lower panel. An equivalent amount of total cell extract from HeLa cells was loaded for each lane and the proteins separated by SDS PAGE, electroblotted and probed both with a monoclonal anti-SMN1 antibody and with an anti-tubulin antibody as a loading control. ( b ) The graph shows average signal intensity and standard deviation for three independent experiments from HeLa cells transiently transfected with U47snoMEN-PR, HBCsnoMENv1-PR and HBCsnoMENv2-PR plasmids, as shown in . The SMN1/GFP-SMN1 signal ratio was normalised to the tubulin signal. Ratios were calculated by comparison with endogenous SMN1 signals in control (No transfection), untransfected cells. Transfection efficiency was determined by counting the number of GFP positive cells in 100 randomly selected cells (TF efficiency: orange bar). P -values are significant according to the Student's t -test; * P <0.05, ** P <0.01, *** P <0.001. ( c ) Graph shows a box plot of GFP-SMN1/DAPI signal ratio comparing transfected U47snoMEN-PR, HBCsnoMENv1-PR and HBCsnoMENv2-PR plasmids. Signals were calculated from 50 randomly selected cells. ( d ) Results of proliferation/cytotoxicity assays comparing control and SMN1 protein replacement cells. A GFP alone expression vector was transiently transfected into HeLa cells as a negative control. The effects of transient transfection with vectors expressing either endogenous SMN1 targeted 47snoMEN, HBCsnoMEN without SMN1 expression (47snoMEN and HBCsnoMEN), or pGFPSMN1-U47snoMEN-PR (47snoMEN-PR), were also measured. Note, both 47snoMEN and HBCsnoMEN showed cytotoxic effects when transiently transfected into HeLa cells, however, this cytotoxicity was rescued by transfection with the 47snoMEN-PR vector. P -values are significant according to the Student's t -test ( P <0.05).

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Expressing, Western Blot, SDS Page, Control, Standard Deviation, Transfection, Comparison, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

doi: 10.1371/journal.pone.0154759

Figure Lengend Snippet: ( a ) Images of protein replacement stable cell lines. Expression of FP proteins was confirmed by fluorescence imaging. Bar is 10 μm. ( b ) Expression levels of endogenous SMN1 and GFP-SMN1 proteins were measured by western blot analysis. An equivalent amount of total cell extract from U2OS and U2OS GFP–SMN1-47PR stable cells was loaded for each lane and the proteins separated by SDS PAGE, electroblotted and probed both with a monoclonal anti-SMN1 antibody and with an anti-tubulin antibody as a loading control. ( c ) Gene-expression profiles were compared between U2OS and U2OS GFP–SMN1-47PR cells by quantitative mass spectrometry analysis. Comparison of protein expression levels detected by mass spectrometry for U2OS and U2OS GFP–SMN1-47PR cells. Each SILAC experiment was independently repeated three times. Correlation between protein ratios of SILAC experiments visualised on a 2D logarithmic graph for all detected proteins, identified as previously described [ , ]. On the x and y axis, log 2 (H/L ratio) correlates with the enrichment in U2OS cells and U2OS GFP–SMN1-47PR cells for experiment 1 and experiment 2, respectively. Graph shows a distribution pattern of plot numbers. SILAC ratio values of labelled proteins are listed in .

Article Snippet: Fluorescent proteins were immunoprecipitated using an anti-GFP monoclonal antibody (GFP–TRAP_A, Chromotek), as previously described [ , ].

Techniques: Stable Transfection, Expressing, Fluorescence, Imaging, Western Blot, SDS Page, Control, Gene Expression, Mass Spectrometry, Comparison, Multiplex sample analysis