anti rnf4 rabbit  (Proteintech)

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) AML cells exhibit an elevated expression of RNF4 (derived from TNMplot.com; based on TGCA data). P value of Mann–Whitney U -test is indicated ( p = 2.22e-21); n = 407 (normal), n = 151 (tumor). Normal: Minima = 77, Maxima = 10,063, Median = 1009, Q1 = 675, Q3 = 1533, Upper whisker = 2726; Tumor: Minima = 691, Maxima = 10,010, Median = 1640, Q1 = 1283, Q3 = 2317.5, Upper whisker = 3750. Dashed lines: 25th and 75th percentile; solid line: 50th percentile (median); Whiskers: range within 1.5×IQR from Q1/Q3. ( B ) Effect of RNF4 expression level on AML patient survival. High expression of RNF4 is associated with a poor prognosis (analyzed using UALCAN). P value of Kaplan–Meier analysis is indicated ( p = 0.041); n = 42 (high expression), n = 121 (low expression). ( C , D ) RNF4 CRISPR dropout experiment in Cas9-expressing OCI-AML3 ( C ) or MV4-11 ( D ) cells. The transduction efficiency of PE-positive cells was around 50%. Survival rate was measured by flow cytometry and normalized to the empty vector control on day 3. Error bars show the standard deviation of the mean, n = 3 (biological replicates). ( E ) Cell viability assay of OCI-AML2 cells 6 days after RNF4 KD with siRNA in combination with decitabine (5-azadC) [1 μM] or aphidicolin [0.075 μg/ml]. P values of two-tailed, unpaired Student’s t -tests are indicated; error bars show the standard deviation of the mean, n = 8 (biological replicates, each three technical replicates). DMSO: p = 0.1754, Decitabine: p < 0.0001, Aphidicolin: p = 0.0532. ( F ) Viability assay of OCI-AML2 cells after RNF4 KD with siRNA in combination with different decitabine concentrations to measure dose-response dependency. IC 50 values were determined by a nonlinear dose-response-inhibition fit, n = 2 (biological replicates). .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY, Whisker Assay, CRISPR, Transduction, Flow Cytometry, Plasmid Preparation, Control, Standard Deviation, Viability Assay, Two Tailed Test, Inhibition

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A , B ) RNF4 CRISPR dropout experiment in Cas9-expressing OCI-AML3 ( A ) or MV4-11 ( B ) cells. The transduction efficiency of PE-positive cells was around 50%. Survival rate was measured by flow cytometry and normalized to the empty vector control on day 3. Error bars show the standard deviation of the mean, n = 3 (biological replicates). ( C , D ) Confirmation of RNF4 KO in OCI-AML3 ( C ) and MV4-11 ( D ) Cas9-expressing cells after transduction with three different guideRNAs by immunoblotting. Cells were transduced and medium was exchanged after 1 day, and supplemented with 2.5 µg/ml puromycin. After 3 days (OCI-AML3) and 4 days (MV4-11), cells were harvested. Tubulin was used as loading control. ( E ) Validation of RNF4 KD corresponding to Fig. (Rep1–Rep8) and 1 F (Rep1–Rep2) by immunoblotting. Cells were harvested 3 days after the performance of KD. Tubulin was used as loading control. Quantification of the RNF4 signal, normalized to tubulin, is indicated.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Plasmid Preparation, Control, Standard Deviation, Western Blot, Biomarker Discovery

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) The RNF4 binder CCW16 (left panel) was utilized for PROTAC development using diverse linkers (central panel) as well as VHL and CRBN E3 ligands (right panel). ( B ) NanoBRET assay for CRBN and VHL E3 ligand-based RNF4 PROTACs to determine cell membrane permeability in intact cells transiently expressing CRBN (left panel) or VHL (right panel) with an N-terminally tagged NanoLuc. Error bars show the standard deviation of the mean, n = 4 (biological replicates). ( C ) Treatment of HeLa WT cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Cells were treated 30 min before PROTAC treatment [5 µM] with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] and harvested after 6 h. Control cells were treated with DMSO. Tubulin was used as loading control. Same experiment is also shown in Fig. . ( D ) HeLa Flag-RNF4 endogenously tagged cells were transfected with His-Ub for 48 h and pretreated with TAK-243 [1 µM], followed by PROTAC 2c [5 µM] or CCW16 [5 µM] treatment for 6 h. Enrichment of ubiquitylated proteins was performed by denaturing Ni-NTA pulldown. Higher molecular weight bands show ubiquitylation signal. Control cells were treated with DMSO. HA-pulldown was performed as a negative control. ( E ) Whole cell proteome analysis by mass spectrometry. HeLa cells expressing RNF4 from a doxycycline-inducible promoter were treated either with CCW16 [5 µM] (upper panel) or 2c [5 µM] (lower panel) for 6 h. Results of the TMT-based MS analysis are visualized in a volcano plot comparing PROTAC treatment vs. DMSO control. Hits considered as significantly upregulated are highlighted in red (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3) and hits considered as significantly downregulated are highlighted in blue (log 2 (ratio) ≤ −0.58, −log 10 ( p value) ≥ 1.3). The identification of those candidates was based on a two-sided Student’s t -test analysis comparing the normalized TMT abundances of CCW16/ 2c treatment with DMSO control treatment. Experiments were performed with four biological replicates. ( F ) Validation of proteomic results by immunoblotting in HeLa WT and HeLa RNF4 KO cells. Same treatment procedure as in ( E ). Tubulin was used as loading control. .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Membrane, Permeability, Expressing, Standard Deviation, Western Blot, Control, Transfection, Molecular Weight, Negative Control, Mass Spectrometry, Biomarker Discovery

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) NanoBRET assay for PROTAC 2b to determine cell membrane permeability in intact and permeabilized cells transiently expressing VHL with an N-terminally tagged NanoLuc. Error bars show the standard deviation of the mean, n = 4 (biological replicates). ( B ) NanoBRET assay for CRBN and VHL E3 ligand-based RNF4 PROTACs to determine cell membrane permeability. IC 50 values of Figs. and EV2A are indicated. ( C ) HeLa WT cells were depleted of RNF4 (siRNF4) or SENP6 (siSENP6) for 72 h or were treated with CCW16-derived PROTACs [5 µM] for 6 h. Control cells were treated with DMSO. RNF4 and SENP6 levels were visualized by immunoblotting. Tubulin was used as loading control. ( D ) Treatment of HeLa FLAG-RNF4 (endogenously tagged) cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Different concentrations and time points are indicated. Control cells were treated with DMSO. Tubulin was used as loading control. ( E ) Evaluation of RNF4 degradation level by immunoblotting in NB-4 cells after treatment with different RNF4-targeting PROTACs. Control cells were treated with DMSO. Vinculin was used as loading control. ( F ) Treatment of OCI-AML2 cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Cells were pretreated with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] 30 min before PROTAC treatment [5 µM] and harvested after 6 h. Control cells were treated with DMSO. Tubulin was used as loading control. ( G ) Evaluation of CRBN and VHL levels in HeLa WT cells by immunoblotting after pretreating cells with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] 30 min before PROTAC treatment [5 µM], followed by harvesting after 6 h. DMSO was used as a control treatment, and tubulin as loading control. Same experiment is also shown in Fig. . ( H ) Whole cell proteome analysis by mass spectrometry. HeLa cells expressing RNF4 from a doxycycline-inducible promoter were treated with 1a [5 µM] for 6 h (same experiment as in Fig. ). Results of the TMT-based MS analysis are visualized in a volcano plot comparing PROTAC treatment vs. DMSO control. Hits considered as significantly upregulated are highlighted in red (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3) and hits considered as significant downregulated are highlighted in blue (log 2 (ratio) ≤ −0.58, −log 10 ( p value) ≥ 1.3). The identification of those candidates was based on two-sided Student’s t -test analysis comparing the normalized TMT abundances of 1a treatment with DMSO control treatment. Experiments were performed with four biological replicates.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Membrane, Permeability, Expressing, Standard Deviation, Derivative Assay, Control, Western Blot, Mass Spectrometry

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Scheme outlining in vitro interaction studies of biotin-CCW16 and GST-based purified RNF4. Biotin-CCW16 modification of RNF4 was visualized by immunoblotting with a streptavidin protein linked to a fluorophore. ( B ) Biotin or biotinylated CCW16 was incubated with wild-type GST-RNF4 or mutants with cysteine residues changed to serines as indicated. Covalent biotin-CCW16-RNF4 conjugates were detected by streptavidin immunoblotting. ( C ) Experimental procedure of in vitro interaction studies of CCW16 or PROTAC 2a with GST-RNF4 WT to identify modified cysteine residues in RNF4 by mass spectrometry. The experiment was performed with three replicates. ( D ) AlphaFold2 (Jumper et al, ; Varadi et al, , ) model of human RNF4, highlighting the CCW16 modified cysteine residue identified in the mass spectrometry experiment in ( C ) in pink, the two catalytic, but unmodified cysteine residues are shown in blue, the not detectable cysteine residue 51 is shown in gray and all remaining cysteine residues are highlighted in green. ( E , F ) Same procedure as in ( B ). GST-RNF4 cysteine to serine mutations are indicated. Covalent biotin-CCW16-RNF4 conjugates were detected by streptavidin immunoblotting. .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: In Vitro, Purification, Modification, Western Blot, Incubation, Mass Spectrometry, Residue

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) MS spectrum of CCW16 modified GST-RNF4 on cysteine residue 91, corresponding to Fig. C, . ( B ) Detected RNF4 peptides including the respective modifications on different cysteine residues (as indicated), corresponding to Fig. C, . ( C ) MS spectrum of 2a modified GST-RNF4 on cysteine residue 91, corresponding to Fig. C, .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Modification, Residue

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Pretreatment of HeLa WT cells with MG-132 [20 µM] 30 min before treatment with CCW28-3 [10 µM] or dBET6 [500 nM] and incubation for 6 h (left immunoblot). Pretreatment of HeLa WT cells or HeLa RNF4 KO cells with MG-132 [20 µM] 30 min before treatment with CCW28-3 [10 µM] or dBET6 [500 nM] and incubation for 6 h (right immunoblot). Both blots show the same experiment. Tubulin was used as loading control. *Unspecific band. ( B ) Quantification of BRD4 levels (short and long isoform) after CCW28-3 treatment in HeLa WT and HeLa RNF4 KO cells. Experiment was performed in three independent replicates. P values of two-tailed, unpaired Student’s t -tests are indicated; error bars show the standard deviation of the mean, n = 3 (biological replicates). Short isoform: WT CCW28-3 vs. DMSO: p < 0.0001, RNF4 KO CCW28-3 vs. DMSO: p = 0.0235, CCW28-3 KO vs. WT: p = 0.1343. Long isoform: WT CCW28-3 vs. DMSO: p = 0.0019, RNF4 KO CCW28-3 vs. DMSO: p = 0.0202, CCW28-3 KO vs. WT: p = 0.4538. ( C ) Treatment of HeLa WT cells with MG-132 [20 µM] or MLN4924 [500 nM] for 30 min followed by treatment with CCW28-3 [10 µM] or dBET6 [500 nM] for 6 h and evaluation by immunoblotting. Tubulin was used as loading control. *Unspecific band. ( D , E ) Measurement of BRD4 levels based on luciferase. Control KD (siControl, ( D )) and RNF4 KD (siRNF4, ( E )) were performed in HEK BRD4-HiBiT cells for 72 h, followed by treatment with different concentrations of CCW16 (upper panel) or CCW28-3 (lower panel) for 4, 6, and 24 h. Luciferase activity was measured by the addition of the large luciferase fragment (largeBiT) and substrate. Error bars show the standard deviation of the mean, n = 4 (technical replicates). ( F ) Confirmation of KD efficiency 3 days after performance of KD of Fig. EV4D,E by immunoblotting. ( G ) Evaluation of cell viability after CCW28-3 treatment in HEK BRD4-HiBiT cell lines by CellTiterGlo assay ( n = 2, biological replicates). Concentrations and time points are indicated.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Incubation, Western Blot, Control, Two Tailed Test, Standard Deviation, Luciferase, Activity Assay

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Volcano plot of quantitative MS analysis after biotin-CCW16 pulldown of HeLa WT cell lysates. Significantly enriched interactors are shown in red (log 2 (ratio) ≥ 1, –log 10 ( p value) ≥ 1.3). Identification of candidates is based on two-sided Student’s t -test analysis comparing LFQ intensities of biotin-CCW16 pulldown and biotin control pulldown. Experiment was performed in biological triplicates. Proteins involved in the reduction of peroxides are additionally highlighted. ( B ) Gene Ontology term enrichment analysis of biological processes (GOBP) of the 38 biotin-CCW16 modified proteins (from Fig. ) identified by MS (log 2 (ratio) ≥ 1, –log 10 ( p value) ≥ 1.3). Shown here are the top ten enriched biological processes. The enrichment analysis was done using the ShinyGO tool, applying an FDR cutoff of 0.05. ( C ) RNF4 immunoblotting of streptavidin pulldown in HeLa WT cells. Same experiment as in Fig. . Tubulin was used as loading control. FT flow through, PD pulldown, h.e. high exposure. ( D ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly upregulated proteins (from Fig. ) identified by MS (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3). Shown here are the top three enriched biological processes. The enrichment analysis was done using the ShinyGO tool, applying an FDR cutoff of 0.05. ( E ) HeLa WT or OCI-AML2 cells were pretreated with ferrostatin-1 [10 µM] followed by treatment with CCW28-3 (10 µM for HeLa WT, 1 µM for OCI-AML2) for 6 h. BRD4 levels were evaluated by immunoblotting. Control cells were treated with DMSO. Tubulin was used as loading control. *Unspecific band. ( F ) Validation of overexpression of human GPX4 WT (hGPX4) compared to empty vector (mock) in HT-1080 cells used in Fig. . β-actin was used as loading control.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Control, Modification, Western Blot, Biomarker Discovery, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: A. Inducible expression of the anti-GFP nanobody (GFPNb) fusion protein. A doxycycline-inducible promoter was used to express the estrogen-binding domain (EBD) fused to the camelid anti-GFP nanobody and the E3 ligase fragment from the Rnf4 ubiquitin ligase. We also produced this fusion protein lacking the EBD moiety. All GFPNb-expressing clones contained a 4X HA tag at the GFP amino-terminus to allow detection of fusion protein expression. B. The theoretical function of the two different components of the GFPNb-directed proteolysis system is depicted. First, a translational fusion between a target protein of interest and GFP is generated in the A. fumigatus genome. This target protein-GFP fusion is susceptible to the activity of the GFPNb-containing fusion protein. The GFP segment of the target protein is recognized by the anti-GFP nanobody with its associated EBD and E3 ligase enzyme Rnf4. Rnf4 in turn recruits an E2 enzyme, ultimately leading to the formation of polyubiquitin chains on the target protein with attendant degradation by the proteasome.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Expressing, Binding Assay, Ubiquitin Proteomics, Produced, Clone Assay, Generated, Activity Assay

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: Whole cell protein extracts were prepared from AfS35 cells (wt) or from AfS35 containing an integrated doxycycline-inducible EBD-GFPNb-Rnf4-expressing clone and a gpdA-controlled GFP gene. Strains were grown in Sabouraud dextrose media in the absence (-) or presence (+) of doxycycline and β-estradiol as indicated. Equal amounts of protein from each extract were resolved by SDS-PAGE and analyzed by western blotting. The transferred membrane was stained with Ponceau S to confirm equal loading followed by visualization with antibodies recognizing the hemagglutinin tag (α-HA) or GFP (α-GFP). Molecular mass standards are indicated on the righthand side of each panel. The numbers at the bottom of lanes 2-4 provide quantitation of the GFP signal relative to that seen in the absence of either doxycycline or β-estradiol.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Expressing, SDS Page, Western Blot, Membrane, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: A strain containing a ncaA -GFP fusion gene was transformed with the doxycycline-inducible GFPNb-Rnf4-producing fusion gene. The untransformed ncaA -GFP strain was used as a control. These strains were grown in Sabouraud dextrose media with or without doxycycline and β-estradiol as indicated at the bottom of the panel. Whole cell protein extracts were prepared and analyzed as described above. Molecular mass standards are shown on the right.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Transformation Assay, Control

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: GFP cassettes were inserted at the carboxy-termini of 3 genes involved in azole susceptibility along with the previously characterized ncaA locus. These genes were cyp51A , abcG1 and atrR . All these strains contained a copy of the doxycycline-inducible EBD-GFPNb-Rnf4-producing fusion gene integrated into their fcyB gene. Appropriate transformants were grown overnight in Sabouraud dextrose media with or without the addition of doxycycline and whole cell protein extracts prepared. Equal amounts of each extract were then analyzed by western blotting as above. Molecular mass standards are indicated and the numbers in each lane represent levels of protein normalized to the expression seen in the absence of doxycycline.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: The doxycycline-inducible EBD-GFPNb-Rnf4-containing strain expressing GFP fusion genes corresponding to Cyp51A-, AbcG1-, NcaA- and AtrR-GFP were tested for their voriconazole phenotypes by a disc diffusion assay. ∼100 conidia were placed on Sabouraud dextrose plates containing (+) or lacking (-) 25 μg/ml doxycycline. A disk containing 1 μg of voriconazole was then placed in the center of each dish and incubated at 37°C for 48 hr. One representative assay of at least 3 trials is shown and the numbers refer to the diameter from the edge of the disk to the start of the growing fungal mat.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Expressing, Diffusion-based Assay, Incubation

Journal: bioRxiv

Article Title: Regulated proteolysis of green fluorescent protein fusion proteins in Aspergillus fumigatus

doi: 10.1101/2025.07.14.664774

Figure Lengend Snippet: A. Strains expressing Erg6-GFP fusion proteins along with doxycycline-inducible forms of either EBD-GFPNb-Rnf4 or GFPNb-Rnf4 were grown in the presence (+) or absence (-) of doxycycline and whole cell protein extracts prepared. Equal amounts of each extract were resolved on SDS-PAGE and analyzed by western blotting using the indicated anti-HA or anti-GFP antisera. Locations of the proteins of interest are denoted by the labeled arrows and molecular mass standards are listed at the left. B. Phenotypes caused by transcriptional repression of Dox off-erg6 or doxycycline-inducible proteolysis of Erg6-GFP were tested on media lacking (-) or containing (+) 25 mg/L doxycycline.

Article Snippet: Initially, the 4X HA-EBD-GFPNb- rnf4 (construct 1) was synthesized by Genscript Inc. and cloned into DraIII-SacII digested pφλfcyB-tetON-sGFP (kindly provided by Fabio Gsaller ( )) to make the plasmid pSP141.

Techniques: Expressing, SDS Page, Western Blot, Labeling