anti herc5 polyclonal antibody  (Proteintech)

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Schematic of HERC5 and its mutants used in this study. HERC5 contains an N-terminal RLD domain (pink) and a C-terminal HECT domain (light green). Numbers shown above each domain indicate amino acid (a.a.) positions. A C-terminal 3×MYC epitope tag was fused to HERC5 WT, ΔRLD, ΔHECT, and C994A expression vectors. ( B ) Schematic of the L1 retrotransposition assay. Left: the retrotransposition-competent L1.3-expressing vector (cepB-gfp-L1.3) contains an enhanced green fluorescent protein retrotransposition indicator cassette ( mEGFPI ) in the 3′ UTR of L1 in the opposite orientation relative to the sense strand of L1 transcription. The mEGFPI cassette is interrupted by an intron in the same orientation relative to L1 transcription. This ensures that EGFP is expressed only after successful retrotransposition. Right: HEK293T cells were transfected with the L1-expressing plasmid containing the mEGFPI indicator cassette. The EGFP-positive cells were quantified using flow cytometry. The figure was created with BioRender. Bottom: retrotransposition efficiency was calculated by normalizing the percentage of EGFP-positive cells obtained in transfection with L1-expressing plasmid (cepB-gfp-L1.3) to that obtained in transfection with an RT-deficient L1-expressing plasmid lacking the intron in mEGFPI (cepB-gfp-L1.3RT[-] intronless). ( C ) L1 retrotransposition assay in HEK293T cells using the pCMV-3Tag-9 vector system. Top: timeline of the assay. HEK293T cells were co-transfected with the L1-expressing vector (cepB-gfp-L1.3) and either pCMV-3Tag-9 (control), HERC5 WT, HERC5 mutants, or MOV10 (positive control). Cells independently co-transfected with cepB-gfp-L1.3RT(-) intronless served as transfection-normalization controls. The transfected cells were selected with blasticidin (10 µg/mL), and the percentage of EGFP-positive cells was determined by flow cytometry. Bottom: L1 retrotransposition assay with HERC5 overexpression using pCMV-3Tag-9 in HEK293T. MOV10 and RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as controls. X-axis, name of the transfected constructs. Y-axis, relative L1 retrotransposition efficiency compared to the control (pCMV-3Tag-9, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of at least three independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( D ) Protein expression levels of HERC5 WT and mutants in HEK293T. Cells were transfected with HERC5 constructs (pCMV-3Tag-9 vector system). HERC5 and GAPDH proteins were detected by western blot using anti-MYC and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. The predicted molecular weights of the proteins are indicated on the right of the blots. ΔRLD showed two bands, likely due to multiple N-terminal ATG start codons. Although the predicted molecular weight of ΔRLD is ∼80 kDa, it migrated below 75 kDa, probably because of its high glutamic acid and aspartic acid content, consistent with a previous observation . ( E ) L1 retrotransposition assay with IFN-α and HERC5 knockdown in HEK293T cells. Top: timeline of the assay. HEK293T cells were treated with IFN-α (100 U/mL) (day −2) and siRNA (day −1) and then transfected with the WT L1-expressing construct (cepB-gfp-L1.3) (day 0). The RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as a negative control. After blasticidin selection (10 µg/mL), the percentage of EGFP-positive cells was measured. Bottom left: L1 retrotransposition assay with IFN-α and siRNA treatments in HEK293T cells. X-axis, siRNA and IFN-α treatments. Y-axis, relative L1 retrotransposition efficiency to the non-targeting siRNA control (siControl) in the absence of IFN-α (set to 1.0). The error bars and p -values were calculated as in (C). Each dot represents an independent biological replicate. Bottom right: the relative retrotransposition ratio calculated by normalizing values obtained with IFN-α to those without IFN-α in the left panel. The ratio of siControl is set to 1.0 to compare with that of siHERC5. The p -values were calculated using a two-tailed, unpaired Student’s t-test; ** p < 0.01. Each dot represents an independent biological replicate. ( F ) Endogenous HERC5 protein expression levels with IFN-α and siRNA treatments in HEK293T cells. HERC5 and GAPDH proteins were detected by western blot using anti-HERC5 and anti-GAPDH antibodies, respectively. GAPDH served as a loading control.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Control, Positive Control, Over Expression, Construct, Western Blot, Molecular Weight, Knockdown, Negative Control, Selection, Two Tailed Test

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A – C ) Protein levels of modified L1 constructs with HERC5. HEK293T cells were co-transfected with modified L1 and HERC5-expressing vectors. The cells were harvested 3 days post-transfection, and protein levels were assessed by western blotting. Top: schematic of modified L1 vectors. Left: schematic of pKN035 (A), which expresses monocistronic ORF1p tagged with a T7 gene 10 epitope. Middle: schematic of pTMO2F3 (B), which expresses monocistronic ORF2p with a 3×FLAG epitope. Right: schematic of pTMF3 (C), which expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. Bottom: the protein expression levels were assessed by western blotting. HERC5 and MOV10 were detected by an anti-MYC antibody. ORF1p, ORF2p, and GAPDH were detected by anti-T7, anti-FLAG, and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. ( D ) Alu retrotransposition assay with full-length L1 in HeLa-HA. Top: HeLa-HA cells were co-transfected with the Alu and full-length L1-expressing construct (pKN040) and either pCMV-3Tag-8-Barr (control), a HERC5-expressing construct, or a MOV10-expressing construct. Cells were selected with G418 (500 µg/mL), stained with crystal violet, and the resulting colonies were counted. The representative images of stained G418-resistant colonies are shown below each condition. The colony numbers of pKN040 were normalized to transfection efficiency and determined as retrotransposition efficiency. MOV10 and the RT mutant served as controls. X-axis, name of the transfected constructs. Y-axis, relative Alu retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of four independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. Bottom: schematic of pKN040. The Alu sequence contains the neomycin-resistant gene cassette in the opposite direction of Alu transcription, and the self-splicing intron was inserted into the neomycin-resistant gene in the same direction as the Alu sequence, together with the full-length L1 sequence. S.D.: splice donor site, S.A.: splice acceptor site. The plasmid figure was created with BioRender. ( E ) Alu retrotransposition assay with monocistronic ORF2 in HeLa-HA. Top: HeLa-HA cells were co-transfected with the Alu and monocistronic ORF2-expressing construct (pTM489) and either pCMV-3Tag-8-Barr, a HERC5-expressing construct, or a MOV10-expressing construct. The assay was conducted as noted in (D). X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). MOV10 and the EN/RT mutant served as controls. The error bars and p -values were calculated as in (D). Bottom: schematic of pTM489, which is similar to pKN040, but does not contain the ORF1 sequence.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Modification, Construct, Transfection, Expressing, Western Blot, Control, Staining, Mutagenesis, Sequencing, Plasmid Preparation

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A–C ) Differential effects of HERC5 on non-human LINE retrotransposition. To measure retrotransposition efficiency, HEK293T cells were co-transfected with either mouse TG F 21 (A), mouse ORFeus-Mm (B), or zebrafish L2-2 (C) together with either the control vector, a HERC5-expressing vector, or a MOV10-expressing vector. Cells were selected with puromycin (1 µg/mL), and the percentage of EGFP-positive cells was determined by flow cytometry. Cells co-transfected with cep99-gfp-L1.3RT(-) intronless served as transfection-normalization controls. X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-8-Barr, set to 1.0). Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. ( D ) Summary of HERC5 effects on retrotransposition. A schematic of retrotransposon structures is shown. Requirements for ORF1 and ORF2p, and whether HERC5 inhibits each element, are indicated for different retrotransposons (human L1.3, mouse L1, zebrafish L2-2, and human Alu ). ( E ) Schematic timeline of the estimated divergence times of small HERC paralogs. HERC4 is predicted to have emerged earliest (>595 million years ago, Mya), HERC3 to have arisen >∼476 Mya, HERC6 ∼430 Mya, and the HERC5 paralog ∼413 Mya. Divergence times are based on the phylogenetic analysis in . The silhouette images of organisms were downloaded from PHYLOPIC version 2.0 ( https://www.phylopic.org/ ). Mice lack HERC5 but have HERC6, which has the E3 ligase activity of ISGylation. Humans have both HERC5 and HERC6; only HERC5 has the activity. ( F ) L1 retrotransposition assay with the small HERC family. HEK293T cells were co-transfected with an L1-expressing construct (cepB-gfp-L1.3) and a member of the small HERC family (HERC4, HERC5, hHERC6, and mHERC6). Cells were selected with blasticidin (10 µg/mL), and the percentage of EGFP-positive cells was measured by flow cytometry. The RT mutant served as a control. X-axis, name of the transfected constructs. Y-axis, relative L1 retrotransposition efficiency compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as noted in (A).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Construct, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Interaction of ORF1p with HERC5 WT and its mutants. Top left: timeline of the experiment. HEK293T cells were co-transfected with L1 and HERC5 expression vectors. Cells were harvested on 4 days post-transfection, and ORF1p-FLAG complexes were immunoprecipitated. Top right: schematic of L1-expressing plasmids. The pJM101/L1.3 plasmid contains the full-length L1.3, and pJM101/L1.3FLAG expresses ORF1p tagged with a FLAG epitope at the carboxyl terminus. Bottom: the input and anti-FLAG IP reactions were analyzed by western blotting. pJM101/L1.3 served as a negative control. HERC5 and its mutants expressed from pEBNA were detected by an anti-MYC antibody, and ORF1p was detected by an anti-FLAG antibody. ( B ) Co-immunoprecipitation of ORF1p and HERC5 with RNase treatment. HEK293T cells were co-transfected with L1 and HERC5 expression vectors. ORF1p-FLAG complexes were purified as in (A) and were treated in the presence or absence of RNase A. The rightmost lane shows the RNase A-treated ORF1p-FLAG complex. HERC5 and ORF1p were detected by anti-MYC and anti-FLAG antibodies, respectively. ( C ) Interaction of the ORF1p RNA-binding mutant (RBM) with HERC5. HEK293T cells were co-transfected with HERC5 and either L1 WT or RBM expression vectors. Top: schematic diagram of L1 RNP with ORF1p WT (yellow) and RBM (purple). Bottom: the input and anti-FLAG IP reactions were analyzed by western blotting. FLAG-tagged ORF1p WT and RBM were immunoprecipitated. HERC5 and ORF1p were detected by anti-MYC and anti-FLAG antibodies, respectively. ( D ) Retrotransposition assay of L1 ORFeus and L1.3. Top: schematic of the pKN039 plasmid, which expresses codon-optimized ORF1p and ORF2p. L1 ORFeus is driven by the CMV promoter and the L1.3 5′ UTR promoter and terminates with the SV40 poly(A) signal sequence. The mEGFPI retrotransposition indicator cassette was inserted into the L1 3′ UTR. Bottom: the RT-deficient L1 (cepB-gfp-L1.3RT[-]) served as a negative control. X-axis, name of the transfected constructs. Y-axis, relative retrotransposition efficiency compared to the control (pCMV-3Tag-9 was set to 1.0 for each L1 construct). The error bars represent the mean ± the standard error of the mean (SEM) of four independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; *** p < 0.001; n.s.: not significant. ( E ) ORF1p levels from L1.3 and L1 ORFeus with HERC5. HEK293T cells were transfected with L1 ORFeus and either the empty vector (pCMV-3Tag-9) or a HERC5-expressing vector. HERC5, ORF1p, and GAPDH were detected by anti-MYC, anti-ORF1p, and anti-GAPDH antibodies, respectively. ORF1p and GAPDH band signal intensities were measured with Empiria Studio. ORF1p signal intensities were normalized to GAPDH intensities to calculate the ORF1p ratio. The signal intensity of L1.3 ORF1p in the control condition was set to 1.0. GAPDH served as a loading control.

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, FLAG-tag, Western Blot, Negative Control, Purification, RNA Binding Assay, Mutagenesis, Sequencing, Construct, Control

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Experimental design for checking L1 RNA and ORF1p levels. Top: schematic of the full-length L1 construct (pTMF3), which expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. The red bar indicates the amplified region for the RT-qPCR primer pair used to measure L1 RNA levels (primer sequences are described in Materials and Methods). Bottom: timeline of the experiment. HEK293T cells were co-transfected with L1 expression constructs and either pCMV-3Tag-9 (control), HERC5, HELZ2 (positive control for RNA levels) or MOV10 (positive control for protein levels). The cells were harvested 3 days post-transfection. L1 RNA levels were measured by RT-qPCR, and ORF1p levels were measured by western blot or flow cytometry. ( B ) L1 RNA levels with HERC5. The T7 primer pair was used to quantify L1 RNA levels, which were normalized to GAPDH RNA levels. X-axis, name of the transfected constructs. Y-axis, relative L1 RNA level compared to the control (pCMV-3Tag-9, set to 1.0). The error bars represent the mean ± the standard error of the mean (SEM) of three independent biological replicates. Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( C ) ORF1p levels with HERC5. HERC5 and MOV10 were detected by an anti-MYC antibody. ORF1p and GAPDH were detected by anti-T7 and anti-GAPDH antibodies, respectively. GAPDH served as a loading control. ( D ) Flow cytometry analysis of ORF1p-EGFP expression. HEK293T cells were co-transfected with pVan583 and either pCMV-3Tag-9, HERC5, or MOV10 expression vector. Top: schematic of the pVan583 plasmid, which expresses EGFP-tagged ORF1p and mCherry-tagged ORF2p and contains the CMV promoter, the 5′ UTR promoter, and the SV40 poly(A) signal sequence for L1 expression. Bottom left and middle panels: relative percentages and median intensities (FITC-A) of ORF1p-EGFP-positive cells, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as in (B). Bottom right: overlaid frequency distribution plot of FITC-A intensity of control (black), HERC5 (red), or MOV10 (green). X-axis, log-scaled fluorescence intensity. Y-axis, cell count. ( E ) Flow cytometry analysis measuring EGFP expression. HEK293T cells were co-transfected with pKN033 and either pCMV-3Tag-9, HERC5, or MOV10. Top: schematic of the pKN033 plasmid, which is derived from pVan583 and expresses EGFP alone. Bottom left and middle panels: relative percentages and median intensities of EGFP-positive cells, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as in (B). Bottom right: overlaid frequency distribution plot of FITC-A intensity of control (black), HERC5 (red), or MOV10 (green). X-axis, log-scaled fluorescence intensity. Y-axis, cell count. ( F ) ORF1p-EGFP levels with HERC5 WT and its mutants. HEK293T cells were co-transfected with pVan583 and either pEBNA, HERC5 WT, or its mutants. The relative cell percentages and median intensities of ORF1p-EGFP-positive cells are shown in the left and middle panels, respectively. X-axis, name of the transfected constructs. Y-axis, relative percentages or median intensities compared to the control (pEBNA, set to 1.0). The error bars and p -values were calculated as in (B).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Construct, Amplification, Quantitative RT-PCR, Transfection, Expressing, Control, Positive Control, Western Blot, Flow Cytometry, Plasmid Preparation, Sequencing, Fluorescence, Cell Counting, Derivative Assay

Journal: bioRxiv

Article Title: The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

doi: 10.1101/2025.09.09.675047

Figure Lengend Snippet: ( A ) Rationale for the cycloheximide (CHX) treatment experiment. CHX treatment stops de novo protein synthesis. Unstable proteins (colored magenta) are reduced by CHX treatment due to protein degradation, while stable proteins (colored green) are not. The figure was created with BioRender. ( B ) Protein levels following CHX treatment. HEK293T cells were co-transfected with pTMF3 and either pCMV-3Tag-9 or a HERC5-expressing vector and treated with 50 µg/mL CHX for the indicated times (0, 2, 4, and 8 h). HERC5, ORF1p, α-tubulin, and RNF138 were detected by anti-MYC, anti-T7, anti-α-tubulin, and anti-RNF138 antibodies, respectively. α-tubulin served as a stable protein control, whereas RNF138 served as an unstable protein control. N.T., non-transfected cells. ( C ) Quantification of the remaining ORF1p levels in the control (blue line) and HERC5 (red line) from (B). ORF1p and GAPDH band signal intensities were measured with Empiria Studio. The ORF1p intensities were normalized to GAPDH intensities, and the relative ORF1p levels were calculated. X-axis, CHX treatment time. Y-axis, the relative ORF1p level compared to the ORF1p level at 0 h (both control and HERC5 were set to 1.0, respectively). Each dot represents an independent biological replicate. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s.: not significant. ( D ) Polysome profiling analysis of control (blue line) and HERC5 (red line). HEK293T cells were co-transfected with L1 and either pCMV-3Tag-9 (control) or a HERC5 expression vector. Cell lysates were subjected to sucrose gradient centrifugation and separated into the indicated fractions. X-axis, fraction number. Y-axis, UV absorbance at 280 nm, showing the distribution of ribosomal subunits (40S and 60S), monosomes (80S), and polysomes. ( E ) RNA enrichment ratio in polysome fractions. L1 (normalized to GAPDH) and GAPDH RNA levels in each sucrose fraction were measured by RT-qPCR, and the ratio of RNA enrichment (HERC5/control) was calculated. The green and orange lines show L1 and GAPDH RNA ratios, respectively. X-axis, ribosome fraction. Y-axis, ratio of RNA enrichment (HERC5/control). The error bars and p -values were calculated as noted in (C). ( F ) RC-L1 RNP formation efficiency with HERC5. HEK293T cells were co-transfected with pTMF3 and either pCMV-3Tag-9, a HERC5-expressing vector, or a MOV10 expression vector. Cells were harvested on 4 days post-transfection, and ORF2p-3×FLAG complexes were immunoprecipitated. Top left: schematic of full-length L1 constructs (pTMF3 and pTMH3). pTMF3 expresses ORF1p tagged with a T7 gene 10 epitope and ORF2p tagged with a 3×FLAG epitope at their carboxyl termini. pTMH3 is similar to pTMF3 but expresses 3×HA epitope-tagged ORF2p and served as a negative control. Bottom left: the input and anti-FLAG IP reactions were analyzed by western blotting. ORF2p and ORF1p were detected by anti-FLAG and anti-T7 antibodies, respectively. HERC5 and MOV10 were detected by an anti-MYC antibody. Right: the input ORF1p/ORF2p ratios (top) and immunoprecipitated ORF1p/ORF2p ratios (bottom), which indicate RC-L1 RNP formation efficiencies. The protein band signal intensities were measured with Empiria Studio. Each ORF1p signal intensity was divided by the respective ORF2p signal intensity, and the resulting ORF1p/ORF2p ratio was calculated. X-axis, name of the transfected constructs. Y-axis, relative ORF1p/ORF2p ratio of input compared to the control (pCMV-3Tag-9, set to 1.0). The error bars and p -values were calculated as noted in (C).

Article Snippet: Human ARRB2 cDNA was cloned into pCMV-3Tag-8 (Agilent Technologies), enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN002_hHERC5-3×FLAG : Human HERC5 cDNA was cloned into pCMV-3Tag-8, enabling its expression with three copies of a FLAG tag at the C-terminus driven by the CMV promoter. pKN003_hHERC5-3×FLAG_ΔRLD : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the RLD domain. pKN004_hHERC5-3×FLAG_ΔHECT : This plasmid is similar to pKN002_hHERC5-3×FLAG, but lacks the HECT domain. pKN005_hHERC5-3×FLAG_C994A : This plasmid is similar to pKN002_hHERC5-3×FLAG, but contains the C994A mutation, therefore expressing ISGylation-deficient HERC5. pKN024_pJM101ORFeus : This plasmid is derived from pJM101/L1.3, but the ORF1- and ORF2 -coding sequences were replaced with the codon-optimized human L1 ORFeus sequence from pDA093 , which was generously provided by Dr. Kathleen Burns (Addgene plasmid #131390). pKN025_hHERC5-3×MYC_pEBNA : Human HERC5 cDNA fused with three copies of a MYC tag sequence in frame to the 3′ end was cloned into pEBNA, enabling its expression driven by the EF1α promoter. pKN026_hHERC5-3×MYC_ΔRLD_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the RLD domain. pKN027_hHERC5-3×MYC_ΔHECT_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but lacks the HECT domain. pKN028_hHERC5-3×MYC_C994A_pEBNA : This plasmid is similar to pKN025_hHERC5-3×MYC_pEBNA, but contains the C994A mutation, therefore expressing ISGylation activity-deficient HERC5. pKN031_hHERC4-3×MYC : Human HERC4 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pKN032_hHERC6-3×MYC : Human HERC6 cDNA was cloned into pCMV-3Tag-9, enabling its expression with three copies of a MYC tag at the C-terminus driven by the CMV promoter. pVan583 : was previously described in ( , ).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Gradient Centrifugation, Quantitative RT-PCR, Immunoprecipitation, Construct, Negative Control, Western Blot

Journal: Reproductive biomedicine online

Article Title: TKTL1: a new candidate gene in non-obstructive azoospermia

doi: 10.1016/j.rbmo.2025.104895

Figure Lengend Snippet: Verification of selected genes by real-time polymerase chain reaction ( CCN2, CSF3, FSD1L, HERC5, HES1, HSPA1B, PTBP2, SMOC1 ) in human testicular primary (hTP) cells with overexpressed TKTL1 gene versus controls in hTP cells. NC, negative control with GFP (green fluorescent protein) expression ( n = 3 experimental replicates); TKTL1, cells with the overexpressed TKTL1 gene ( n = 3 experimental replicates); WT, wild type cells in in-vitro culture medium only ( n = 3 experimental replicates), Data presented as mean + SD. P -values are presented on each graph. Data analysed by one-way analysis of variance with Tukey’s multiple comparisons test.

Article Snippet: The Human Protein Atlas data showed that HERC5 expression level is highest in spermatogonia.

Techniques: Real-time Polymerase Chain Reaction, Negative Control, Expressing, In Vitro

Journal: Reproductive biomedicine online

Article Title: TKTL1: a new candidate gene in non-obstructive azoospermia

doi: 10.1016/j.rbmo.2025.104895

Figure Lengend Snippet: Verification of selected genes by real-time polymerase chain reaction ( CCN2, CSF3, FSD1L, HERC5, HES1, HSPA1B, PTBP2, SMOC1 ) in testicular tissue from patients with non-obstructive azoospermia (NOA) and controls. Control, male gonadal tissue from men with normal spermatogenesis ( n = 2 technical repeats); NOA TKTL1 , patient with NOA without mutation in the TKTL1 gene ( n = 2 technical repeats); NOA TKTL1 mut , patient with NOA with mutation in the TKTL1 gene ( n = 2 technical repeats). Data presented as mean + SD. P -values are presented on each graph. Data analysed by one-way analysis of variance with Tukey’s multiple comparisons test.

Article Snippet: The Human Protein Atlas data showed that HERC5 expression level is highest in spermatogonia.

Techniques: Real-time Polymerase Chain Reaction, Control, Mutagenesis

Journal: Biology direct

Article Title: E3 ligase HERC5-catalyzed UGDH isgylation promotes SNAI1-mediated tumor metastasis and cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s13062-025-00622-1

Figure Lengend Snippet: Fig. 4 HERC5 enhances the stability of SNAI1 mRNA by inducing UGDH phosphorylation. (A). Protein interaction network of human HERC5, as determined by BioGRID. Many HERC5-interacting proteins, including ISG15 and UGDH, were identified. (B). Schematic illustration of UGDH to promote EMT by increas ing the stability of SNAI1 mRNA, as reported by Nature. 2019;571:127–131 [18]. UGDH, UDP-glucose 6-dehydrogenase. HuR, Hu antigen R. UDP-Glc, uridine diphosphate-glucose. UDP-GlcUA, UDP-glucuronic acid. (C). UGDH protein abundance, phosphorylation level at tyrosine 473, and ratio of Phospho.Y473/ Total UGDH in head and neck cancer (HNC) and normal paracancerous tissues by Cancer Proteogenomic Data Analysis Site (cProSite). (D). Immunoprecipita tion of tyrosine-phosphorylated proteins in lysates from OSCC and para-OSCC tissues, using a pan-phospho-Tyrosine antibody. Western blot analysis was performed using an anti-UGDH antibody to assess tyrosine phosphorylation levels of UGDH in OSCC vs. para-OSCC tissues. (E). SNAI1 mRNA expression lev els in 8 paired OSCC and para-OSCC tissues of our independent hospital cohorts. (F). SNAI1 protein expression levels in 8 paired OSCC and para-OSCC tissues from our independent hospital cohorts. P, para-OSCC tissues. T, OSCC tissues. (G) Relative mRNA levels of SNAI1 in SCC9 (left) or CAL27 (right) cells. (H) SCC9 (left) or CAL27 (right) cells were treated with actinomycin D (1 μg/ml). Remained SNAI1 mRNA levels after treatment were examined at the indicated time points. (I). Immunoprecipitation of tyrosine-phosphorylated proteins in lysates from SCC9 (left) or CAL27 (right) cells, using an anti-pan-phospho-Tyrosine antibody. Western blot analysis was performed using an anti-UGDH antibody to assess tyrosine phosphorylation levels of UGDH. (J) Remained SNAI1 mRNA levels in SCC9 cells after treatment were examined at the indicated time points. (K). SCC9 cells stably overexpressing HERC5 were transiently transfected with pcDNA 3.1 (+) plasmids of a C-terminally Flag-tagged UGDH (wild type or Tyr473Phe (Y473F) mutant type). After 48 h, the remaining SNAI1 mRNA levels were examined at the indicated time points. Data in (G, H, J, K) were expressed as mean values ± SD. #/*/& p < 0.05, ##/**/&& p < 0.01 and ###/***/&&& p < 0.001

Article Snippet: The membranes were treated with blocking buffer (SW3010, Solarbio), followed by incubation in a 1:1000 dilution of indicated primary antibodies: anti-HERC5 antibody (22692-1-AP, ProteinTech), anti-E-cadherin antibody (AF0131, Affinity, Cincinnati, OH, USA), antiVimentin antibody (AF7013, Affinity), anti-cleaved PARP antibody (AF7023, Affinity), anti-γ-H2AX Ser139 antibody (AF3187, Affinity), anti-SNAI1 antibody (AF6032, Affinity), anti-UGDH antibody (sc-137058, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag antibody (66008-4-Ig, ProteinTech), anti-Myc antibody (AE070, ABclonal [1:10000 dilution]), and anti-GAPDH antibody (60004-1-Ig, ProteinTech [1:10000 dilution]) overnight at 4 °C.

Techniques: Phospho-proteomics, Quantitative Proteomics, Western Blot, Expressing, Immunoprecipitation, Stable Transfection, Transfection, Mutagenesis

Journal: Biology direct

Article Title: E3 ligase HERC5-catalyzed UGDH isgylation promotes SNAI1-mediated tumor metastasis and cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s13062-025-00622-1

Figure Lengend Snippet: Fig. 5 HERC5 increases phosphorylation level of UGDH by ISGylation. (A). The cascade reactions of ISGylation. The covalent modification of ISG15 requires an E1 activating enzyme (UBE1L), an E2 conjugating enzyme (UBCH8) and one of three E3 ligases (such as HERC5). Finally, ISG15 links to the Lys (K) resi due of the target protein and mediates a ubiquitin-like modification. (B). Co-IP results. Whole-cell extracts (WCE) from SCC9 (up) or CAL27 (down) cells were subjected to immunoprecipitation with an antibody against HERC5 or a control IgG. Bound proteins were detected by western blot analysis. (C). Heatmap using the CCLE dataset for mRNA expression of ISG15 and its conjugation enzymes, including E1 enzyme UBE1L and E2 enzyme UBCH8, in five OSCC cells (CAL27, SAS, SCC25, SCC4, and SCC9). (D). Immunoprecipitation of tyrosine-phosphorylated proteins in lysates from SCC9 (left) or CAL27 (right) cells, using an anti-ISG15 antibody. Western blot analysis was performed using an anti-UGDH antibody to assess ISGylation of UGDH. (E&F). SCC9 cells were transiently transfected with pcDNA3.1-HA-ISG15, pcDNA3.1-Flag-UGDH and pcDNA3.1-Myc-HERC5 (wild type or Cys994Ala mutant type) plasmids. After 48 h, WCE were subjected to immunoprecipitation with an antibody against HA (E) and pan-phospho-Tyrosine (F). Bound proteins were detected by western blot analysis. (G). SCC9 cells were transiently transfected with pcDNA3.1-HA-ISG15, pcDNA3.1-Myc-HERC5 and pcDNA3.1-Flag-UGDH (wild type or Tyr473Phe mutant type) plasmids. After 48 h, WCE were subjected to immunoprecipitation with an anti-pan-phospho-Tyrosine antibody. Bound proteins were detected by western blot analysis

Article Snippet: The membranes were treated with blocking buffer (SW3010, Solarbio), followed by incubation in a 1:1000 dilution of indicated primary antibodies: anti-HERC5 antibody (22692-1-AP, ProteinTech), anti-E-cadherin antibody (AF0131, Affinity, Cincinnati, OH, USA), antiVimentin antibody (AF7013, Affinity), anti-cleaved PARP antibody (AF7023, Affinity), anti-γ-H2AX Ser139 antibody (AF3187, Affinity), anti-SNAI1 antibody (AF6032, Affinity), anti-UGDH antibody (sc-137058, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag antibody (66008-4-Ig, ProteinTech), anti-Myc antibody (AE070, ABclonal [1:10000 dilution]), and anti-GAPDH antibody (60004-1-Ig, ProteinTech [1:10000 dilution]) overnight at 4 °C.

Techniques: Phospho-proteomics, Modification, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Expressing, Conjugation Assay, Transfection, Mutagenesis

Journal: Biology direct

Article Title: E3 ligase HERC5-catalyzed UGDH isgylation promotes SNAI1-mediated tumor metastasis and cisplatin resistance in oral squamous cell carcinoma.

doi: 10.1186/s13062-025-00622-1

Figure Lengend Snippet: Fig. 6 SNAI1 depletion inhibits HERC5 OE-induced cell invasion and cisplatin resistance. (A&B). A shRNA targeting HERC5 mRNA (shSNAI1) and non- targeting control shRNA (shCtrl) was inserted in pRNA-H1.1/Neo plasmid. The plasmids were transfected into SCC9 cells. After 48 h, expression of SNAI1 mRNA (A) and protein (B) were detected by real-time PCR and western blot analyses. (C&D). Transwell matrigel invasion assays in SCC9 cells. Representa tive photomicrographs (C, Scale bars: 100 μm) and quantification (D) of the invasive cells (stained with crystal violet) at 24 h in lower surface of Transwell chamber. (E). 48 h after transfection, SCC9 cells were treated with 6 μM of cisplatin. Apoptosis of SCC9 cells after 48 h of cisplatin treatment was deter mined by flow cytometer using Annexin V-PE/7-AAD assay. Data in (A, D, E) were expressed as mean values ± SD

Article Snippet: The membranes were treated with blocking buffer (SW3010, Solarbio), followed by incubation in a 1:1000 dilution of indicated primary antibodies: anti-HERC5 antibody (22692-1-AP, ProteinTech), anti-E-cadherin antibody (AF0131, Affinity, Cincinnati, OH, USA), antiVimentin antibody (AF7013, Affinity), anti-cleaved PARP antibody (AF7023, Affinity), anti-γ-H2AX Ser139 antibody (AF3187, Affinity), anti-SNAI1 antibody (AF6032, Affinity), anti-UGDH antibody (sc-137058, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag antibody (66008-4-Ig, ProteinTech), anti-Myc antibody (AE070, ABclonal [1:10000 dilution]), and anti-GAPDH antibody (60004-1-Ig, ProteinTech [1:10000 dilution]) overnight at 4 °C.

Techniques: shRNA, Control, Plasmid Preparation, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Flow Cytometry