Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a – e All samples were transfected with plasmids encoding UBE1L and Myc-tagged UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP and/or FLAG-HERC5. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblotting with anti-Xpress or anti-HERC5 antibody. b Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Myc-EFP, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Myc or anti-Xpress antibody. c HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or its catalytically inactive mutant FLAG-HERC5-CA, alone or in combination. Cell lysates were subjected to NTA pull-down (PD: NTA) under denaturing conditions followed by western blotting with anti-FLAG or anti-Xpress antibody. d Cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, Myc-EFP-WT, or Myc-tagged catalytically inactive EFP mutant Myc-EFP-CS, alone or in combination. Cell lysates were subjected to NTA pull-down, as in c . e HEK293 cells were transfected for 48 h with nonspecific control scrambled siRNA (NC), HERC5 -siRNA (H5), or plasmid encoding Xpress-His-CHIP or FLAG-ISG15, alone or in combination. Cell lysates were subjected to NTA pull-down as in b . f Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Flag-HHARI, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Flag or anti-Xpress antibody. Asterisk indicates IgG heavy chains, and closed arrowhead indicates predicted or right size band

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Control

Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a , b All samples were transfected with plasmids encoding UBE1L and Myc-UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding HA-CHIP, FLAG-ISG15-GG, or FLAG-ISG15-AA, alone or in combination. Cells were treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-ubiquitin antibody. Tubulin served as a loading control. b HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or FLAG-HERC5-CA, alone or in combination, and treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-ubiquitin antibody. Actin served a as loading control. c A549 cells were treated for 48 h with vehicle (−) or IFN-α (1000 U/ml). Immunoprecipitation of cell lysates was performed with preimmune IgG or anti-CHIP antibody, followed by western blotting with anti-ubiquitin or anti-CHIP antibody. Tubulin served as a loading control

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control

Journal: Cell Stem Cell

Article Title: Chromatin and Single-Cell RNA-Seq Profiling Reveal Dynamic Signaling and Metabolic Transitions during Human Spermatogonial Stem Cell Development

doi: 10.1016/j.stem.2017.09.003

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-HERC5; dilution: 1:400 , Atlas Antibodies , cat# HPA043929, RRID: AB_10962492.

Techniques: Immunofluorescence, Histopathology, Methylation Sequencing, Software

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct

Journal: iScience

Article Title: Cellular targets and lysine selectivity of the HERC5 ISG15 ligase

doi: 10.1016/j.isci.2024.108820

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-HERC5 , Thermo Fisher Scientific , CAT# 703675; RRID: AB_2784598.

Techniques: Recombinant, Transfection, CRISPR, Ubiquitin Proteomics, Peptide Fractionation, Mass Spectrometry, Expressing, Plasmid Preparation, Software

Journal: Open Biology

Article Title: Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

doi: 10.1098/rsob.160193

Figure Lengend Snippet: HERC5 is an essential E3 ISG15 protein ligase of parkin. ( a ) HEK293 cells were mock transfected or transfected with expression vector encoding HA-tagged parkin. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-HERC5 or anti-HA antibody. ( b ) HEK293 cells were transfected with expression vector encoding HA-parkin, pcDNA-UBE1 L or Myc-tagged UbcH8 alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-HERC5 or anti-HA antibody. ( c,d ) Where specified, HEK293 cells were transfected with expression vector encoding FLAG-tagged wild-type HERC5 (FLAG-HERC5-WT) ( c ) or its catalytically inactive form (FLAG-HERC5-C994A) ( d ). Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-HERC5, anti-HA or anti-FLAG antibody. ( e ) HEK293 cells were transfected with expression vector encoding HA-parkin, FLAG-ISG15-WT, FLAG-HERC5-WT or FLAG-HERC5-C994A alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody. ( f ) HEK293 cells were mock transfected (−) or transfected for 48 h with non-specific control siRNA (nc; 200 nM) or the indicated concentration of HERC5 -specific siRNA (si- HERC5 ). Cell lysates were immunoblotted with anti-HERC5 antibody. ( g ) HEK293 cells were transfected for 48 h with nc siRNA (50 nM) or si- HERC5 (H5; 50 nM). Where indicated, cells were additionally transfected for 24 h with vector encoding HA-parkin or FLAG-ISG15-WT alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody. ( h ) HEK293 cells were transfected with vector encoding HA-parkin, FLAG-ISG15-WT, Myc-EFP-WT or Myc-EFP-C13/16S alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody. All samples were also transfected with vector encoding FLAG-ISG15-WT, UBE1 L, Myc-UbcH8 and HA-parkin ( c , d ) or UBE1 L and Myc-tagged UbcH8 ( e , g , h ). Expression of transiently transfected proteins was monitored by immunoblotting of cell lysates with anti-HERC5, anti-HA, anti-Myc or anti-FLAG antibody. Actin and Hsp90 served as loading controls. Asterisks indicate IgG heavy chains.

Article Snippet: Polyclonal anti-HA (PAB0861) and anti-HERC5 (H00051191-A01) antibodies were obtained from Abnova (Tebu, France), and monoclonal anti-HA antibody (MMS-101P) was purchased from Covance (Dedham, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Control, Concentration Assay

Journal: Open Biology

Article Title: Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

doi: 10.1098/rsob.160193

Figure Lengend Snippet: Identification of parkin ISGylation sites. ( a ) Schematic diagram of full-length (FL) parkin and its truncated mutants. The parkin ISGylation regions are outlined by the red box. The blue and red ‘K's represent the three lysine residues located in the 291–380 amino acid region of parkin. The two red ‘K's indicate the exact ISGylation sites. ( b , c ) HEK293 cells were transfected with constructs encoding Myc- ( b ) or HA-tagged ( c ) parkin-FL or its truncated mutants (i.e. parkin 81–465 , parkin 226–465 , parkin 291–465 or parkin 381–465 ), or FLAG-ISG15-WT alone or in combination. Cell lysates were immunoprecipitated with anti-Myc ( b ) or anti-HA ( c ) antibodies, followed by immunoblotting with anti-FLAG ( b ), anti-parkin ( c ) or anti-Myc antibody. Red, blue ( c,d ) and white ( c ) asterisks indicate double (2×)-, single (1×)-ISG15-conjugated and unmodified parkin, respectively. Red underlined asterisks indicate predicted double-ISG15-conjugated parkin, and blue underlined asterisks indicate predicted single-ISG15-conjugated parkin ( c ). ( d ) HEK293 cells were transfected with constructs encoding HA-parkin-WT or its point mutants (HA-parkin-K299R, HA-parkin-K349R and HA-parkin-K369R) or FLAG-ISG15-WT alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG or anti-HA antibody. ( e ) HEK293 cells were transfected with vector encoding Myc-parkin-WT or Myc-parkin-K349R/K369R mutant (2KR) and FLAG-ISG15-WT alone or in combination. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-FLAG or anti-Myc antibody. ( f ) HEK293 cells were transfected with vector encoding HA-parkin-WT, HA-parkin-2KR or FLAG-HERC5 alone or in combination. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-HERC5 or anti-HA antibody. Expression of transiently transfected proteins was monitored by immunoblotting of cell lysates with anti-Myc, anti-HA or anti-FLAG antibody. All samples were also transfected with UBE1 L and HA-tagged ( b , e ) or Myc-tagged UbcH8 ( c , d , f ). Actin, Hsp90 and tubulin served as loading controls. Black asterisks indicate IgG heavy chains ( b , d , e ).

Article Snippet: Polyclonal anti-HA (PAB0861) and anti-HERC5 (H00051191-A01) antibodies were obtained from Abnova (Tebu, France), and monoclonal anti-HA antibody (MMS-101P) was purchased from Covance (Dedham, MA, USA).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Expressing