EMBO/soda-nel huggingface dataset:
Genetic control of pluripotency epigenome determines differentiation bias in embryonic stem cells (2021)
Figure 5

Figure 5: trans-QTL regulate chromatin binding of transcription factors required for pluripotency and differentiation.

A. Scatterplot of difference in chromatin accessibility in the 33 BXD inbred versus difference in H3K4me3 modification in the parental strains for trans-caQTL (LOD ≥ 8). ATAC-seq signal represents the log2 ratio of the average signal based on the genotype at the QTL.

B. H3K4me3 level is predicted by the genotype at the QTL versus local genotype. H3K4me3 ChIP-seq for B6 and D2 parental lines compared to two BXD strains (mean ± SEM). Genotypes for both the location of the peak and location of the QTL are indicated below.

C. Scheme to determine whether local (peak) or distal (QTL) genotype predicts chromatin state. A ratio of ~ 1 (log2(ratio) ≈ 0) indicates similar chromatin modification.

D. Log2 fold change of H3K4me3 level when comparing BXD75 or BXD87 to the parental genotype either at the location of the peak or based on the genotype at the QTL for all trans-caQTL for which the local genotype and QTL genotype differ (LOD score ≥ 7).

E. Grey - Publicly available ESC ChIP-seq datasets were evaluated for significant overlap of QTL targets (Fisher's exact test, top ten factors with q-value cutoff < 0.01). To validate a subset of these results ChIP-seq was performed for TRIM28 (N = 3), POU5F1 (N = 2), and P300 (N = 2) in B6 (blue) and D2 (orange) ESCs. Each circle represents a biological replicate or independent ChIP-seq accession from Cistrome.

F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.

G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61).

H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64).

I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.

J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66).

K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51).

Data information: P-values in E and H represent two-sided t-test, * < 0.05, ** < 0.01, **** < 0.0001)
Genetic control of pluripotency epigenome determines differentiation bias in embryonic stem cells (2021)
Figure 6

Figure 6: Validation of Chr 4 QTL on gene expression and chromatin accessibility implicate KRAB-ZFPs in trans regulation.

A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.

B. Gene expression (Riok3 and Rmc1) or chromatin accessibility (18:12.157 Mb) level measured in B6, D2, and (BxD)F1 hybrid mESCs. For this locus, both gene expression and chromatin accessibility are dominantly repressed in the F1 (mean ± SEM)

C. Scatterplot of the additive versus dominant effect for each Chr 4 caQTL target locus. Points along the diagonal lines indicate dominant effects (red - caQTL targets found at the Riok3/Rmc1 locus.

D. MA-plot of significant (FDR < 0.01) differential H3K9me3 level between wildtype (B6) mESCs and those with a 2.47 Mb deletion encompassed by the Chr 4 QTL (n = 2 replicates). Regions that overlap Chr 4 caQTL targets are indicated in magenta (Chr18:12.157 Mb locus from A in yellow).

E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).

F. LOLA analysis of significant enrichment between sites that change between WT and Chr 4 KO mESCs and overlap QTL hotspot targets. For H3K4me3, H3K4me1, and H3K27ac there was only one ChIP experiment performed and therefore all sites with a log2 fold-change ≥ 1 were used for overlap enrichment.

G. Box and whisker plot indicating direction of change for histone modifications between WT and Chr 4 KO cells.

H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.

I. Model of trans-regulation of chromatin state for the Chr 4 QTL. Local variation at the Chr 4 QTL results in differential expression of KZFPs, leading to decreased expression of distal genes through recruitment of TRIM28 and formation of heterochromatin (H3K9me3).

Data information: In the boxplot, the central lines represent medians, the box represents the first and third quartile, and the whiskers extend 1.5 times the interquartile range.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 1

Figure 1. Analysis of BYDV 17K protein phosphorylation in vivo and in vitro.

Detection of phosphorylated 17K in BYDV-infected cells using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. dpi, days after BYDV inoculation; P, phosphorylated 17K bands; λ-PP, λ-phosphatase.

Active HvSnRK1, but not its total amount, was increased in BYDV-infected barley plants as compared to the mock controls. P-SnRK1 or SnRK1, enzymatically active SnRK1 (with T172 phosphorylation) or unphosphorylated SnRK1 protein.

C,D Protein-protein interactions between 17K and HvGRIK1 (C) or HvSnRK1 (D) revealed using Y2H assays.

Analysis of 17K phosphorylation by HvGRIK1-HvSnRK1 kinases in vitro. The kinase reactions were set up by mixing the desired GST fusions of 17K, 17K3A, 17K5A, HvGRIK1, and HvSnRK1 kinase domain (KD), with the reaction products analyzed using Phos-tag SDS-PAGE-immunoblotting assays with an anti-17K antibody. P, phosphorylated 17K bands; arrowhead, unphosphorylated 17K.

A diagram showing BYDV 17K residues phosphorylated by HvGRIK1 alone or by HvGRIK1-HvSnRK1 cascade. The phosphorylated 17K residues were identified using LC-MS/MS experiment with higher than 91% of 17K amino acid covered in MS spectra.

Schematic representation of the dephosphomimic (17K3A and 17K5A) and phosphomimic (17K5D) mutants of BYDV 17K used in this study.

Data information: The datasets shown were reproducible in at least three independent experiments.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 2

Figure 2. Overexpression of HvSnRK1 increases 17K phosphorylation and promotes barley defense to BYDV.

Confirmation of the expression of SnRK1K139R-YFP or SnRK1-YFP fusion protein each in two transgenic lines, SnRK1K139R-YFP-L13 and -L22 or SnRK1-YFP-L4 and -L17, by immunoblotting. The protein samples analyzed here or in (B) and (C) were all prepared from BYDV-infected barley plants at 21 dpi.

Phosphorylation of BYDV 17K was increased in SnRK1-YFP-L4 plants overexpressing SnRK1-YFP but reduced in SnRK1K139R-YFP-L13 ectopically expressing SnRK1K139R-YFP compared with WT control. P, phosphorylated 17K bands.

BYDV disease symptoms were substantially attenuated in the transgenic lines overexpressing SnRK1-YFP, but aggravated in those ectopically expressing SnRK1K139R-YFP compared with WT control.

D-F Quantitative comparisons of plant height (D), maximum root length (E), and total root length (F) among the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).

G,H Transcript (G) and protein (H) levels of viral CP gene in the set of BYDV-infected barley materials shown in (C) (using the samples collected at 21 dpi).

Data information: In (D-G), data are presented as means ± SD of 30 (D-F) or 4 (G) plants for each line, with those labeled by different letters being significantly different. P < 0.001 (D-F); P < 0.05 (G), ANOVA and LSD for multiple comparisons. The datasets displayed in (A-C and H) were all representative of three independent experiments.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 4

Figure 4. Effects of phosphorylation on the VSR activity of BYDV 17K as analyzed using phosphomimic (17K5D) and dephosphomimic (17K5A) mutants.

The VSR function of 17K5D was drastically decreased compared to 17K and 17K5A, as assessed using the 16c transgenic tobacco. The P19 VSR of tomato bushy stunt virus and an empty plasmid vector (35S::EV) were used as positive and negative controls, respectively. The GFP fluorescence was imaged at 4 days post agroinfiltration of the indicated constructs.

Levels of GFP siRNA detected by sRNA blotting for the panel of tobacco materials shown in (A), with arrowhead indicating the main band of GFP siRNA.

A diagram illustrating four recombinant pea early browning virus (PEBV) vectors, with the RNA2 modified to express GFP, 17K, 17K5A, or 17K5D. The original RNA2 genes, 29.8K and 23K (Goulden et al, 1990; Constantin et al, 2004), were replaced by the coding sequence of GFP, 17K, 17K5A, or 17K5D in the recombinant viruses shown. The five PEBV genes, including 30K on RNA1 and CP on RNA2 are depicted.

Transcript levels of CP in the tobacco plants infected by four recombinant PEBVs at 21 dpi. CK, uninfected plants.

Protein levels of PEBV CP and corrected expression of 17K, 17K5A or 17K5D in the panel of tobacco plants shown in (D), revealed using immunoblotting with anti-PEBV CP or anti-17K antibody.

Coverage depth of vsiRNAs along PEBV genomic RNA1 obtained for the plants infected by PEBV-17K5D, PEBV-GFP, or PEBV-17K. sRNA sequencing was conducted at 14 dpi with three biological repeats.

Data information: In (D), data are means ± SD (n = 4 biological replicates), with those labeled by different letters being significantly different (P < 0.05, ANOVA and LSD for multiple comparisons). The datasets displayed in (A, B, and E) were typical of three independent experiments.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 5

Figure 5. 17K5D interacts with HvSDN1 and binds BYDV vsiRNA.

A,B 17K5D, but not BYDV 17K, interacted with HvSDN1 in Y2H (A) and LC (B) assays. Expression of 17K and 17K5D in the LC assays were verified by immunoblotting using anti-17K antibody.

C 17K5D, but not 17K or 17K5A, showed binding to BYDV vsiRNA. The GST fusions of 17K, 17K5D, and 17K5A, as well as free GST, were used in the RNA EMSA assays with biotin-labeled vsiRNA899 or vsiRNA899 duplex as probes.

Data information: The experiments shown were all repeated three times with similar results obtained.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 6

Figure 6. 17K5D impairs vsiRNA cleavage by recombinant HvSDN1 in vitro.

Cleavage of BYDV vsiRNA899 by HvSDN1 (His-HvSDN1), with AtSDN1 (His-AtSDN1) serving as a positive control. Asterisk denotes cleavage products. The GST fusions of 17K and 17K5D did not show vsiRNA cleavage activity.

Inhibition of HvSDN1-catalyzed vsiRNA cleavage by GST-17K5D but not GST-17K. Free GST protein served as a negative control. Asterisk denotes cleavage products.

C,D The three mutants of 17K5D (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) retained the ability to interact with HvSDN1 as revealed using LC assays (C), but their GST fusions failed to bind vsiRNA899 (D). Neither GST nor GST-17K showed vsiRNA899 binding.

A fluorogenic substrate for quantitatively analyzing the enzymatic activity of HvSDN1. The 5´ and 3´ends of vsiRNA899 were labeled by 5-carboxyfluorescein (R) and carboxytetramethylrhodamine (Q), respectively. Cleavage of such labeled vsiRNA substrate by HvSDN1 would remove the quenching activity of Q, thus allowing the emitted fluorescence to be recorded by suitable detectors.

F-H Quantitative analysis of HvSDN1 cleavage activities, with the fluorescence recorded using Typhoon FLA9500 (F and G) or Roche LC 480 platform (H). Compared with free GST or GST-17K, the GST fusions of 17K5D or three derivative mutants (17K5Dm4, 17K5Dm2a, and 17K5Dm2b) inhibited HvSDN1-catalzyed cleavage of fluorogenic vsiRNA899, which was revealed by a fluorospot assay (F), relative comparison of fluorescence values (G), or comparison of the absolute amounts of vsiRNA cleaved by HvSDN1 (H).

Data information: In (G and H), data are means ± SD (n = 3 independent assays), with those labeled by different letters being significantly different (P < 0.05, ANOVA and LSD for multiple comparisons). The datasets displayed in (A, B, and D) were reproducible in three separate experiments.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 7

Figure 7. P17K impedes the degradation of HvAGO1-associated vsiRNA by HvSDN1 in BYDV-infected barley.

Protein-protein interactions detected for 17K5D and HvAGO1, 17K and HvAGO1, or HvSDN1 and HvAGO1 using Y2H assays. Both 17K5D and 17K interacted with HvAGO1 through binding to its PAZ and Piwi domains. HvSDN1 interacted with HvAGO1 through the PAZ domain.

Co-IP assays revealing the presence of vsiRNA-containing HvAGO1-HvSDN1-P17K complex in BYDV-infected WT, SnRK1K139R-YFP-L13, and SnRK1-YFP-L4 plants, with mock-inoculated SnRK1-YFP-L4 individuals as controls. The immunoprecipitates, prepared using anti-HvAGO1 antibody, were analyzed for the presence of HvSDN1 (by immunoblotting with anti-HvSDN1 antibody), P17K (asterisk) and 17K (arrowhead) (using Phos-tag SDS-PAGE coupled immunoblotting), and BYDV vsiRNA (by sRNA blotting). Compared with WT control, the HvAGO1 immunoprecipitates derived from SnRK1-YFP-L4 contained higher levels of P17K and vsiRNA but less amount of HvSDN1, whereas the reverse was observed for SnRK1K139R-YFP-L13.

Co-IP assays demonstrating that the interaction between HvAGO1 and HvSDN1 was weakened by P17K. The immunoprecipitates were prepared using anti-HvSDN1 antibody from the panel of plants shown in (B). Relative to WT control, the HvSDN1 immunoprecipitates derived from SnRK1-YFP-L4 contained much less HvAGO1, whereas the opposite was observed for SnRK1K139R-YFP-L13.

17K5D compromised the interaction between HvSDN1 and the PAZ domain of HvAGO1. The LUC signal resulted from the interaction between HvSDN1-HvAGO1 PAZ domain was significantly decreased in the presence of 17K5D but not that of 17K.

Data information: In (B and C), the relative band intensities were obtained using ImageJ software. In (D), data are means ± SD (n = 9 biological replicates). **, P < 0.01 (Student's t-test). The datasets shown in (A-C) were all representative of three independent assays.
Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection (2022)
Figure 8

Figure 8. Genetic analysis of the function of 17K5D or SDN1 in regulating anti-BYDV RNAi in common wheat.

Verification of 17K5D expression in the transgenic lines 17K5D-L3 and -L7 by immunoblotting with anti-17K antibody.

B-D Comparison of BYDV disease symptoms including plant growth (B), plant height (C), and maximum root length (D) between two transgenic lines and WT control at 21 dpi.

E,F The transcript (E) and protein (F) levels of viral CP gene in the BYDV-infected samples shown in (B).

BYDV vsiRNA abundance in the set of samples shown in (B), as detected by sRNA blotting at 14 dpi.

CRISPR/Cas9-induced mutation in the TaSDN1-D homoeolog of common wheat. A single nucleotide (arrowed) was deleted in the target region (written in red), resulting in a premature stop codon (marked in blue). Selfing TaSDN1-Dd produced the homozygous mutant (TaSDN1-dd) and the plant line (TaSDN1-DD) homozygous for WT TaSDN1-D.

Relative to TaSDN1-DD line, TaSDN1 protein was decreased in TaSDN1-Dd and TaSDN1-dd mutants, with a stronger reduction found for TaSDN1-dd. The reactive TaSDN1 protein detected in TaSDN1-dd was caused by the un-mutated TaSDN1-A and -B homoeologs.

J,K Quantitative comparison of plant height (J) and maximum root length (K) among TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi of BYDV.

L,M Relative transcript (L) and protein (M) levels of BYDV CP gene in TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at 21 dpi.

BYDV vsiRNA abundance in TaSDN1-DD, TaSDN1-Dd, and TaSDN1-dd plants detected by sRNA blotting at 21 dpi of BYDV.

The morphologies of BYDV-infected TaSDN1-DD, TaSDN1-Dd and TaSDN1-dd plants at adult stage, which were photographed at 20 days post anthesis. The lower panel illustrates the grain yield per plant obtained for BYDV-infected TaSDN1-DD, TaSDN1-Dd or TaSDN1-dd.

Data information: In (F, G, I, M, and N), the relative band intensities were obtained using ImageJ software. In (C-E and J-L), data are means ± SD of 15 (C and D), 8 (E), 30 (J and K), or 4 (L) plants for each line, with those marked by different letters are significantly different (P < 0.05, ANOVA and LSD for multiple comparisons). The datasets shown in (A, B, F, G, I, and M-O) were all representative of three independent experiments.
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 1

Figure 1. USP36 promotes SUMOylation in cells

(A, B). Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB.

(C-E). Knockdown of USP36 reduces SUMOylation in cells. H1299 (C), HeLa (D) and U2OS (E) cells infected with scrambled (scr) or USP36 shRNA encoding lentiviruses were assayed by IB.

(F). USP36 promotes nucleolar SUMOylation. H1299 cells transfected with control or V5-USP36 were fractionated to the cytoplasm (Cyto), nucleoplasm (Np) and nucleolus (No) fractions and assayed by IB.

Data information: SUMO2/3 conjugates are indicated in all the top panels.
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 2

Figure 2. The N-terminus of USP36 is necessary for promoting SUMOylation

(A). H1299 cells were transfected with Flag-USP36 or the indicated deletion mutants and assayed by IB. FL, full length.

(B). Schematic diagram of USP36 indicating that the N-terminus of USP36, which contains the USP catalytic domain, is required for its SUMO E3 activity. Catalytic residues C131 and H382 are shown.

(C). H1299 cells transfected with WT USP36, the C131A, or H382A mutant were assayed by IB to detect SUMOylated proteins.

(D). H1299 cells transfected with WT USP36 or the indicated mutants and V5-SUMO2 were assayed by IB using anti-V5 antibody to detect total protein SUMOylation.

(E). H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 3

Figure 3. USP36 interacts with Ubc9 and SUMO and mediates SUMOylation in vitro.

(A). USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB.

(B). USP36 interacts with Ubc9 in vitro. Purified His-Ubc9 was incubated with GST, GST-USP361-800 or GST-USP361-420. Bound protein was detected by IB. GST and GST-fusion proteins were shown in the bottom panel by coomassie staining.

(C). USP36 interacts with SUMO in cells. H1299 cells transfected with either control or Flag-USP361-800 were assayed by co-IP using anti-Flag, followed by IB with anti-SUMO2/3 antibodies.

(D). USP36 interacts with SUMO2 chains in vitro. Recombinant SUMO2 chains were incubated with GST or indicated GST-USP36 fragments. Bound proteins were assayed by IB using anti-SUMO2/3 antibodies. Coomassie staining of the recombinant proteins was shown in bottom panel.

(E). USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 4

Figure 4. USP36 SUMOylates Nop58 and Nhp2 in cells and in vitro

(A, B). H1299 cells transfected with His-SUMO2 and/or USP36 were subjected to Ni2+-NTA agarose beads pulldown (PD) followed by IB to detect SUMOylation of Nop58 (A) and Nhp2 (B). *indicates a non-specific anti-Nhp2 antibody-reacting band.

(C). USP36 promotes Nop58 and Nhp2 SUMOylation in the nucleolus. Nucleoli isolated from HeLa cells transfected with His-SUMO2 and/or USP36 were subjected to Ni2+-NTA PD, followed by IB.

(D, E). Knockdown of USP36 reduces Nop58 and Nhp2 SUMOylation in cells. Cells infected with scr or USP36 shRNA lentiviruses were assayed by IB. The SUMOylated Nop58 (D) and Nhp2 (E) are indicated.

(F-H). USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 5

Figure 5. USP36 interacts with snoRNPs and promotes snoRNP protein group SUMOylation.

(A, B). USP36 promotes SUMOylation of DKC1 and Nop56. H1299 cells transfected with indicated plasmids were subjected to Ni2+-NTA PD followed by IB to detect SUMOylated Nop56 and DKC1 respectively. The protein expression was shown in the bottom of each panel.

(C, D). USP36 interacts with both box C/D (C) and H/ACA (D) snoRNP complexes. H1299 cells transfected with empty vector or Flag-USP36 plasmid were assayed by IP with anti-Flag antibody, followed by IB to detect the indicated proteins.

(E). USP36 interacts with snoRNP proteins in the nucleolus. The nucleoli purified from H1299 cells transfected with empty vector or Flag-USP36 plasmid were assayed by co-IP with anti-Flag antibody, followed by IB to detect the indicated proteins.

(F). Half-life assays. The WCL from H1299 cells treated with 50 μg/ml cycloheximide (CHX) for indicated time were assayed by IB for the indicated snoRNP proteins.

(G). USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.