EMBO/soda-nel huggingface dataset:
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 4

Rap2b negatively regulates the autophagic response induced by the toxin.

(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments. (C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments. (D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 5

Rap2b knockdown does not affect Hla-induced autophagy.

(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments (n = 50 cells/condition). (C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods. Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test). (D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments. (E) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 6

cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.

(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 7

Calpain inactivation hampers the inhibitory effect of dbcAMP.

(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments. (C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 8

cAMP inhibits <italic>S. aureus</italic>–induced autophagy.

(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments. (C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 9

EPAC overexpression suppresses the autophagic response induced by <italic>S. aureus</italic>.

CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (upper panel) or myc-ΔEPAC (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. Images are representative of three independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta upon the infection. These data are representative of three independent experiments. (C) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S. aureus. ** p<0.01 (paired Student's t-test, n = 20 cells/condition). These data are representative of three independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 10

Rap2b inhibits the autophagy induced by <italic>S. aureus</italic>.

(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments. (B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments. (C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experiments.
cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response. (2012)
Figure 11

Active Rap2b is decreased to allow the autophagy response induced by Hla and <italic>S. aureus</italic>.

(A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments. (B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway. (2015)
Figure 1

Inflammation-induced AID expression does not contribute to carcinogenesis

A–C AID expression was analyzed in colon and pancreatic human cell lines and in pancreas explants from C57BL/6 mice. Samples were treated as indicated with 50 ng/ml TNF-α. (A) qRT–PCR analysis of AID expression in LoVo and SW480 colon cell lines. n = 5 (LoVo); 3 (SW480). **P-value: LoVo: 0.0017; SW480: 0.0079. (B) qRT–PCR analysis of AID expression in AsPC and PaTU-8988S pancreatic cell lines (n = 2). *P-value: AsPC: 0.0369; PaTU-8988S: 0.0119. (C) qRT–PCR analysis of AID expression in pancreatic explants from wild-type mice (n = 3). *P = 0.0242.

D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histologic inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.

Data information: All data are mean values ± SEM. Statistical differences were analyzed by two-tailed unpaired Student’s t-test.
AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway. (2015)
Figure 2

Heterologous AID expression does not promote carcinoma development

Schematic of the constructs used for conditional expression of AID in epithelial cells. An AID-IRES-GFP cassette preceded by a transcriptional STOP flanked by LoxP sites was introduced by homologous recombination within the endogenous Rosa26 locus (R26AID+/KI mice, top). R26AID+/KI mice were bred with Villin-CRE and p48-CRE mice to achieve specific AID expression in colon and pancreas, respectively (bottom).

GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm.

qRT–PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.

Kaplan–Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).
AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway. (2015)
Figure 3

AID expression in pancreas promotes DNA lesions

A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009.

C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.

D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.

Data information: Statistical differences were analyzed by two-tailed unpaired Student’s t-test.
AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway. (2015)
Figure 4

AID expression in pancreas promotes proliferation and NKG2D ligand expression

Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266.

Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom.

Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black).

Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012.

Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.

Data information: Statistical differences were analyzed by two-tailed unpaired Student’s t-test.
AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway. (2015)
Figure 5

AID expression in pancreas promotes immune infiltration and cell death

Hematoxylin–eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098.

CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149.

Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm.

TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392.

Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.

Data information: Statistical differences were analyzed by two-tailed unpaired Student’s t-test.
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 1

Fig. 1. Deletion of HRD1 specifically in liver leads to growth retardation

(A) GO functional analysis of the differential genes in the fasted and refed condition.

(B, C) Body weight of the WT and L-HRD KO mice at the age of 12 weeks. (n=5 for each group)

(D) Body heights and tibia lengths of the WT and L-HRD KO mice. (n=5 for each group)

(E) Phosphorylation of STAT5 in WT and HRD1 LKO liver.

(F, G) Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group).

Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 2

Fig. 2. Genetic ablation of hepatic HRD1 elevated hepatic mRNA and circulating Fgf21.

(A) Volcano plot of differential genes between WT and HRD1 LKO livers.

(B) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice. (n=5 for each group).

(C) Serum FGF21 protein levels in the WT and L-HRD1 KO mice. (n=5 for each group).

(D) Correlation analysis of the gene expression (Red: Upregulated genes and Blue: Downrelated genes) between HRD1 LKO mice and FGF21 transgenic mice.

(E) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L-HRD KO mice. (n=10 for each group)

(F) Examples of ovarian histology from WT and HRD1 LKO mice.

(G) Representative examples of estrus cycles in WT and L-HRD1 KO mice as determined by vaginal cytology.

(H) Percentage of feeding, water intake and activity in the light time. (n=8 for each group).

(I) H&E stain of white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding.

(J) Ucp1 and Dio2 mRNA levels of adipose tissue from (I) (n=5 for each group).

Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 3

Fig. 3. 737 candidate HRD1 substrates are identified by protein level enrichment (A) Flow chart for the RNA-Seq and proteomic identification of differential mRNA and proteins. (B) Volcano plot of differential proteins between WT and HRD1 LKO livers. (C) Venn blot of the overlaps of the differential genes in mRNA and protein levels. (D) GO functional analysis of the differential proteins from proteomic screening. (E) Overlap of the increased genes identified between RNA-Seq and proteomics. (F) Protein levels of IRE1α, NRF1, RXRβ, NOTCH1, SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition. (n=6 for each group).
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 4

Fig. 4. HRD1 ERAD decrease the stability of CREBH through mediating its ubiquitination

(A, B) Hepatic CREBH protein (A) and mRNA (B) levels in the WT and L-HRD1 KO mice. (n=6 for each group).

(C) Western Blot analysis CREBH protein stability after HRD1 over-expression.

(D) Western Blot analysis of hepatic CREBH and HRD1 protein levels in WT and HRD1 LKO mice. The mice were fasted 16 hours and refed for another 4 hours.

(E) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag-agarose in transfected HEK293T.

(F) Endogenous interaction between CREBH and HRD1 in liver.

(G) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag-agarose in transfected HEK293T.

(H) Hepatic CREBH ubiquitination level in the WT and L-HRD1 KO mice from (D).

(I) Relative protein levels of CREBH, HRD1 and ubiquitination of CREBH.

(J) Primary hepatocytes were isolated from WT and HRD1 LKO mice and Western Blot analysis CREBH protein stability.

(K) Full-length and C-terminal of HRD1 were generated.

(L) Interactions of full-length and C-terminal of HRD1 with CREBH were measured by Co-IP.

Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.
Hrd1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination (2018)
Figure 5

Fig. 5. HRD1 ERAD increase CREBH ubiquitination at the lysine residue 294 through C-terminal RING-finger and proline rich domain (A) Full-length and activated form of CREBH was generated. (B) Interactions of full-length and activated form of CREBH with HRD1 were measured by Co-IP. (C, D) Ubiquitination of CREBH and CREBH K294R were measured after co-transfected C-terminal of HRD1. (E) Western Blot analysis CREBH or CREBH-K294R protein stability after HRD1 protein over-expression. (F) Western Blot analysis of the CREBH WT or K48 only ubiquitination level after HRD1 C-terminal co-expression. (G) Schematic presentation of wild type Ub and its mutants. (H) Western Blot analysis of the CREBH K6, K11, K27, K29 and K33 only ubiquitination level after HRD1 co-expression. (I) Western Blot analysis of the CREBH WT, K27R ubiquitination level after HRD1 full-length co-expression. (J) Western Blot analysis of the CREBH WT, K27 and K48 only ubiquitination level after HRD1 C-terminal co-expression. Data information: The data are representative of three independent experiments (mean ± s.d.).