EMBO/SourceData huggingface dataset:
Figure 4(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.
Figure 5Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf The indicated P-values were determined by unpaired, two-tailed Student's t-test.
Figure 6(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies. (B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.
Figure 7(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates. Bars are labelled with the number of replicates.(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****). Bars are labelled with the number of replicates.
Figure 8E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).
Figure 2A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.ChIP analyses using anti-Rpb3 (B) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).ChIP analyses using anti-SUMO (C) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).D qRT-PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed Error bars represent the SD from three RNA samples. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).ChIP assays using anti-Rpb3 antibodies in ulp2Δ cells Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes , while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01ChIP assays using anti-SUMO (F) antibodies in ulp2Δ cells ;Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from three (F) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).
Figure 3A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strainsC ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05).D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.E Chromatin association assays using strains After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies.F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.
Figure 4A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.C Mutant htb1-K123R ulp2Δ and htb1-K123R ULP2 strains with a pRS316-ULP2 cover plasmid were grown on the indicated plates at 30°C for 2-3 days. Three double mutant isolates were tested in parallel.D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.
Figure 5A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti-Flag blotting for Flag-H2B was used to verify the similar loading.C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays. Data information: Asterisks indicate significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01).F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading.ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01).
Figure 6A The ratio of CTD S2-P relative to Rpb3 at 3' ORF regions was analyzed by ChIP using anti-S2-P and anti-Rpb3 antibodies. The error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).B A ulp2Δ single mutant and three isolates of ulp2Δ ctk1Δ with YCplac33-ULP2 were grown on SD-Ura or SD+5-FOA plates at 30°C for 2-3 days.E ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).F Co-IP analysis of Ctk1 and histone H2B interaction. WT and ulp2Δ strains expressing the indicated tagged proteins were precipitated with IgG-Sepharose. IP and INPUT proteins were subjected to immunoblotting with PAP (Ctk1-TAP) and anti-Flag (Flag-H2B) antibodies. Protein signals were quantified relative to PGK and then normalized to the WT value.G ChDIP of Flag-H2B-SUMO in the indicated strains The "Pro" and "ORF" of UTH1 and ATP2 genes indicate positions 1 and 2 of PCR fragments, respectively The error bars indicate the SD from three independent ChDIPs. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).H Sensitivity to 6-AU of indicated htb1-1 htb2-1 (null alleles for both chromosomal H2B genes) strains carrying pRS314 plasmids that express Flag-H2B or 2SUMO-H2B.I, J ChIP analyses of the indicated strains Occupancy of Rpb3 (I) and the ratio of CTD S2-P relative to Rpb3 (J) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).K ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).L Immunoblot analysis of TAP-tagged Ctk1 proteins in extracts prepared from the strains Anti-PGK blotting shows similar loading.M, N ChIP analyses of the indicated strains Occupancy of Rpb3 (M) and the ratio of CTD S2-P relative to Rpb3 (N) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).
Figure 1(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.(E) Intracellular cytokine staining assay of human TNF-α and IL-2 in sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for 6 hours (average frequency of positive staining YFP+ cells in sh-NC cells without stimulation = 1).(F) ELISA analysis of IL-2 and IL-6 production in the culture supernatants of human PBMCs transfected with si-NC or si-p38IP, and then stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 (αCD3/CD28) for 24 hours.(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.
Figure 2(A) Immunoblot analysis with the indicated antibodies of total cell lysates of sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times. Actin served as a loading control (throughout).(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.
Figure 3(A) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography-tandem mass spectrometry. m/z, mass/charge ratio.(B) HEK293T cells were transfected with YFP-p38IP, and the cell lysates aliquots were immunoprecipitated with IgG, anti-TAB1, anti-TAB2 and anti-TAK1 respectively, and then immunoblotted with the indicated antibodies.(C) sh-NC or sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times and lysed, followed byTAK1 immunoprecipitation. TAK1 beads were washed three times with lysis buffer and two times with kinase buffer, then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST-IKKβ(111-250aa) protein as substrate to perform in vitro kinase assay, and then immunoblot analysis with p-IKKβ antibody.(D) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAB2 and then immunoblotted with anti-TAK1 and anti-TAB2. Expression of cMyc-p38IP was determined in the immunoblotted lysates with anti-cMyc.(E) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAK1 and then immunoblotted with anti-TAB2, anti-TAB1, anti-HA and anti-TAK1. Expression of HA-p38IP was determined in the immunoblotted lysates with anti-HA.(F) sh-NC and sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and the cell lysates were immunoprecipitated with anti-TAB2 and immunoblotted with anti-TAK1 and anti-TAB2.(H) Jurkat TAg T cells transfected with cMyc vector or cMyc-p38IP were stimulated with 10μg/ml anti-CD3 for the indicated times, and then were lysed and immunoprecipitated with anti-TAK1 antibody and immunoblotted with anti-K63Ub, anti-cMyc and anti-TAK1 antibodies. Lysates were blotted with the indicated antibodies, and actin served as the loading control.(I) sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-K63Ub and anti-TAK1. Lysates were immunoblotted with the indicated antibodies.(J) Jurkat TAg T cells transfected and stimulated as in (H) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2.(K) sh-NC and sh-p38IP Jurkat E6.1 cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2. Data (B-F, H-K) are representatives of at least three independent experiments.
Figure 4(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph.(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.
Figure 5(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).
Figure 6(B) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.(E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.(F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies.(G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting.(H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).