Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Representative IHC images of FFPE human normal, low grade or high grade dyplasia cervical or HN oropharyngeal tissues, as indicated, stained with anti-UBC9 antibody. Scale bars = 200nm. (B) Representative IHC images FFPE human normal, dysplastic or tumoral (squamous cell carcinoma-SCC) HN oropharyngeal HPV positive or negative tissues, as indicated, stained with anti-UBC9 antibody. Scale bars = 200nm. Samples were classified as HPV positive by DNA and RNA testing, as described in . (C) Box and whisker plots showing the median and 10–90 percentiles of percentage of UBC9 positivity in HPV negative and HPV positive dysplastic and tumoral (SCC) HN oropharyngeal specimens. n = 20 (HPV negative) and 57 (HPV positive) different tissues. Outliers are shown by black circles. *P<0.05, **P < 0.001; ***P < 0.0001(one-way ANOVA followed by Tukey post hoc test). (D) Representative IHC images of FFPE human LSIL cervical tissue stained with anti-UBC9 or anti-Ki-67 antibodies, as indicated. Scale bars = 200nm.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Staining, Whisker Assay

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) and (B) Left: Representative IHC images of FFPE human normal, LSIL or HSIL cervical tissues stained with anti-SUMO1 or anti-SUMO2/3 antibodies, respectively. Right: Percentage of SUMO1-, or SUMO2/3-positive pixels, respectively, in FFPE human cervical tissues. Scale bars = 200nm. Data are expressed as percentage of positive plus strong positive stained pixels. The black lines represent the mean for each group. n = 37 (normal), 20 (LSIL), 20 (HSIL) different patients. *P<0.05; **P < 0.001; ***P < 0.0001(one-way ANOVA followed by Bonferroni post hoc test). (C) Representative IHC images of a FFPE human cervical tissue stained with anti-UBC9, anti-SUMO1 or anti-SUMO2/3 antibodies, respectively. (D) Top: Correlation between UBC9 and SUMO1 expression in paired normal (left) or lesion (right) tissues. n = 39 (normal), 38 (lesions) respectively. Bottom: Correlation between UBC9 and SUMO2/3 expression in paired normal (left) or lesion (right) tissues. n = 41 (normal), 38 (lesions) respectively. Spearman rank correlation was applied to analyze association between variables. Scattered plots with linear regression lines, Spearman r correlation coefficient, and p value are reported. (E) Representative IHC images of a FFPE human cervical tissue co-stained with anti-UBC9 (brown) and anti-SUMO1 (red). Note that almost all cells were both UBC9 and SUMO1 positive.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Staining, Expressing

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) and (B). Left: Representative WB of HKs transduced with HPV E6/E7 from the indicated HPV types or SV40 large T antigen recombinant retroviruses. n = 4 different biological replicates. p53 accumulation is reported to check SV40 large T antigen expression. (C) Left: Representative WB analysis of HPV16 E6/E7 HKs after transfection with scramble (siLuc) or two different Ubc9 siRNAs. Middle: Representative cytometric dot plot of Annexin V/PI staining of siLuc or siUbc9s HPV16 E6/E7 HKs. y axes: Annexin V-FITC staining; x axes: PI staining. Quadrants of early, late and necrotic cells are reported. Right: Flow cytometric analysis of siLuc or siUbc9s cells apoptosis. Data are expressed as percentage of early and late apoptotic cells. Bars represent means ± SEM of n = 10 different biological replicates. **P < 0.001; ***P < 0.0001 (two-way ANOVA followed by Bonferroni post hoc test) compared to siLuc cells. (D) Left: Representative WB analysis of HaCaT cells after transfection with control (empty) or UBC9 protein. Middle: Representative cytometric dot plot of Annexin V/PI staining of empty or UBC9-HaCaT. y axes: Annexin V-FITC staining; x axes: PI staining. Quadrants of early, late and necrotic cells are reported. Right: Flow cytometric analysis of empty or UBC9-HaCaT apoptosis. Data are expressed as percentage of early and late apoptotic cells. Bars represent means ± SEM of n = 4 different replicates. *P < 0.05; (two-way ANOVA followed by Bonferroni post hoc test) compared to empty cells.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Transduction, Recombinant, Expressing, Transfection, Staining

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Left: Representative WB of HKs transduced with the indicated recombinant retroviral vectors and analyzed by immunoblotting using anti- UBC9 antibody. Right: quantification of the protein bands intensities, standardized to GAPDH levels. Data are expressed as fold over the empty-transduced HKs. Bars represent means ± SEM of at least n = 9 biological replicates. *P<0.05; **P < 0.001; ***P < 0.0001(Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test). p53 and pRb are reported as control for E6, E7, and mutants expression, respectively. Asterisks mark the phosphorylated forms of pRb. (B) Representative WB of HFs transduced with the indicated retroviral vectors and analyzed by immunoblotting using anti- UBC9, -p53, pRb antibodies. GAPDH and Vinculin are reported as loading control. n = 3 biological replicates. (C) Representative WB of Caski cells transduced with shScramble or shp53, as indicated, and treated with control siRNA (siLuc) or with siE6/E7 as described in Materials and Methods. Samples were analyzed by immunoblotting using anti- UBC9 and anti-p53antibodies. GAPDH is reported as loading control.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Transduction, Recombinant, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Left: Representative WB of empty- or HPV16 E6/E7-transduced HKs treated with vehicle or MG132. Right: normalized UBC9 expression. Data are expressed as fold over the untreated empty-transduced HKs. Bars represent means ± SEM of n = 5 different biological replicates. ns: not significant (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) compared to vehicle control groups. (B) Left: Representative WB of HaCaT cells treated with vehicle or the indicated proteasome inhibitors. Right: normalized UBC9 expression. Data are expressed as fold over untreated cells. Bars represent means ± SEM of n = 6 different biological replicates. ns: not significant (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) compared to vehicle control groups.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Immunolocalization of UBC9 in ultrathin sections of HKs transduced with empty or HPV16 E6/E7 vectors. Original view (left) and higher magnification (right) of boxed regions. Gold particles are selectively enriched in autophagic structures highlighted by arrows. Scale bar = 1 μm. (B) Top: Representative WB of HaCaT cells treated with the indicated autophagic activators (left) and inhibitors (right). Activation of autophagy was monitored by the conversion of LC3 (LC3-I) to the lipidated LC3 (LC3-II) form, a marker of autophagosome production induced by autophagic stimuli . LC3-II accumulation was used to verify autophagic impairment . Bottom: normalized UBC9 expression. Data are expressed as fold over untreated cells. Bars represent means ± SEM of n = 4 different biological replicates. ns: not significant (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) compared to vehicle control groups. ( C) Representative WB analysis of U-2 OS or MCF7 cells treated with chloroquine. n = 3 different biological replicates. (D) Representative WB analysis of MCF7 cells transduced with scramble or ATG5 shRNA. The observed ATG5 band represents the ATG5-ATG12 conjugated form. LC3-I accumulation is reported to evidence autophagic deficiencies promoted by shATG5. n = 3 replicates of a single transduction.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Transduction, Activation Assay, Marker, Expressing, shRNA

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Immunolocalization of LC3 in ultrathin sections of HKs transduced with empty or HPV16 E6/E7 vectors. Original view (left) and higher magnification (right) of boxed regions. Nanogold particles are selectively enriched in autophagic structures highlighted by arrows. Scale bar = 1 μm. (B) Top: Confocal microscopy images of empty or HPV16 E6/E7 transduced HKs immunostained with antibody against LC3 (red). Nuclei were stained with DAPI (blue). Scale bar = 5 μm. Bottom: Quantification of endogenous LC3 dot total area. Data are expressed as number of LC3 dots. Bars represent means ± SEM of n = 50 fields of three different biological replicates. ***P < 0.0001 (independent-sample t test). (C) Left: Representative WB analysis of LC3 and p62 in empty- or HPV16 E6/E7-transduced HKs. Right: LC3 and p62 normalized expression. Data are expressed as fold over empty HK. Bars represent means ± SEM of n = 18 different biological replicates. **P < 0.001 (one-sample t-test). (D) Left: Representative WB analysis of empty- or HPV16 E6/E7-transduced cells treated with vehicle or bafilomycin for 8 hours. Right: normalized p62 expression as fold over empty cells. Bars represent means ± SEM of n = 10 different biological replicates. **P < 0.001 (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test). (E) Left: Representative WB analysis of LC3 and p62 in empty-, E6-, E7-, or HPV16 E6/E7-transduced HKs. Right: LC3 and p62 normalized expression. Data are expressed as fold over empty HK. Bars represent means ± SEM of n = 8 different biological replicates. ns: not significant, *P<0.05, **P < 0.001 (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) (F) Left: Representative WB analysis of UBC9 and p62 expression HKs transduced with the indicated combination of HPV16 E6 wt or mutants and E7 wt. n = 3 biological replicates.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Transduction, Confocal Microscopy, Staining, Expressing

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) Left: Confocal microscopy images of empty or HPV16 E6/E7 transduced HKs treated with vehicle or bafilomycin for 24 hours, and immunostained with antibody against UBC9 (green) and LC3 (red). Nuclei were stained with DAPI (blue). Colocalized dots images show UBC9/LC3 dots colocalization as determined by the Green and Red Puncta Colocalization plugin. Inset show higher magnification views of boxed regions. Arrows highlight colocalized dots. Scale bar = 5 μm. Right: Quantification of UBC9/LC3 colocalization expressed as percentage of colocalized UBC9 with LC3 (double labeled dots) dots on total UBC9 (single labeled plus double labeled dots) per cell, as determined with the Green and Red Puncta Colocalization plugin. Bars represent means ± SEM from at least 47 fields of three different biological replicates. ***P < 0.0001; ns: not significant (one-way ANOVA and Bonferroni post hoc test). (B) Representative WB analysis of UBC9 expression in cells treated as in (A). n = 3 different biological replicates. (C) Top: Representative WB analysis of empty or HPV16 E6/E7 HKs cultured in growth or in starvation medium. LC3-II disappearance was used to confirm autophagy activation . Bottom: UBC9 normalized expression reported as fold over cells grown in normal medium. Bars represent means ± SEM of n = 7 different biological replicates. *P<0.05; ns: not significant (Kruskal–Wallis one-way ANOVA with Dunn’s post hoc test) compared to normal medium-grown cells. (D) Left: Representative IHC images of FFPE human normal, LSIL or HSIL cervical tissues stained with anti-p62 antibody. Right: Percentage of p62 positive pixels in FFPE human cervical tissues. Scale bars = 200nm. Data are expressed as percentage of positive plus strong positive stained pixels. The black lines represent the mean for each group. n = 18 (normal), 11 (LSIL), 18 (HSIL) different patients. ***P < 0.0001 (one-way ANOVA and Bonferroni post hoc test). (E) Correlation between p62 and UBC9 expression human cervical tissues. n = 29 different patients. Scattered plots with linear regression line, Spearman r correlation coefficient, and p value are reported.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Staining, Labeling, Expressing, Cell Culture, Activation Assay

Journal: PLoS Pathogens

Article Title: Autophagy regulates UBC9 levels during viral-mediated tumorigenesis

doi: 10.1371/journal.ppat.1006262

Figure Lengend Snippet: (A) In normal conditions, UBC9 protein expression is retained at physiological levels by basal autophagy in HKs. (B) However, the presence of HPV16 E6/E7 impairs autophagosome-autolysosome fusion. As a consequence, UBC9 is no longer efficiently cleared by the autophagic machinery and accumulates in HPV16 E6/E7 transduced cells. Finally, the increased UBC9 expression leads to extended life-span of HPV16 E6/E7 HKs.

Article Snippet: The following antibodies were used: anti-LC3 (Novus Biologicals), anti-UBC9 (BD Biosciences), anti-LAMP2 (Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: ZNF33B enhanced SUMO conjugation to JEV NS5. ( A ) Immunoblot analysis of JEV NS5 SUMOylation from HEK293T cells co-transfected with UBC9-V5, NS5-FLAG, and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( B–D ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( E and F ) Immunoblot analysis of lysates from HEK293T and SK6 cells infected with JEV, followed by DMSO or the SUMOylation inhibitor 2-D08 (0–200 μM) treatment for 6 h. ( G and H ) The viral titration analysis of the supernatant in JEV-infected cells treated with 2-D08 was conducted by plaque assay. The statistical analysis of JEV titer in 2-D08-treated cells. ( I ) Immunoblot analysis of UBC9 in HEK293T cells transfected with ZNF33B-MYC, followed by JEV infection. The expression of ZNF33B was assessed by measuring the band grayscale with the “ImageJ” software. ( J ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG, pEYFP-SUMO1, and UBC9-V5, followed by treatment with 2-D08 (200 µM). The lysates were subjected to precipitation using anti-HA antibodies for the enrichment of SUMOylated proteins and subsequently probed with specific antibodies to confirm the conjugation of SUMO1 to NS5. ( K–M ) Immunoblot analysis of the effect of ZNF33B and its truncations on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and ZNF33B-HA, ZNF33B ZFs-HA, or ZNF33B ΔZFs-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. All experiments were conducted in triplicate, and data are represented as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test (** P < 0.01 and *** P < 0.001).

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Conjugation Assay, Western Blot, Transfection, Expressing, Software, Infection, Titration, Plaque Assay

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: Lysine residues 269 and 846 are critical for ZNF33B-promoted NS5 SUMOylation. ( A ) The diagram illustration of SUMOylation site prediction from Advanced Services of GPS-SUMO 2.0 ( https://sumo.biocuckoo.cn/online.php ), JASSA version 4 ( http://www.jassa.fr ), and SUMOplot Analysis Program ( https://www.abcepta.com/sumoplot ) websites. The overlap of predicted sites is shown in the bottom table. ( B–D ) Immunoblot analysis of NS5 SUMOylation in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, ZNF33B-MYC, and the WT or mutant constructs of NS5-FLAG, with arginine replacement of the lysine residues at positions 269 ( B ), 287 ( C ), and 846 ( D ). The lysates were subjected to precipitation using anti-GFP antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( E–G ) Immunoblot analysis of the K269R ( E ), K287R ( F ), and K846R ( G ) mutations on the association of ZNF33B with JEV NS5 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-MYC and NS5-FLAG. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( H ) Confocal microscope observation of the colocalization of ZNF33B with the WT or mutations of NS5 in HEK293T cells transfected with ZNF33B-HA and NS5-FLAG with arginine replacement of the lysine residues at positions 269, 287, and 846. Scale bar, 2 µm. ( I ) Left, sequence alignment of SUMO interaction motifs (SIMs) derived from some E3 SUMO enzymes or flaviviruses NS5 proteins, with the dotted line indicating potential SIM. Right, a diagram depicting the SIMs generated by WebLogo ( https://weblogo.berkeley.edu/logo.cgi ). ( J ) The interaction model of JEV NS5 SIM and SUMO1 was generated by AlphaFold Server. The putative interacting amino acids were identified and annotated using PyMOL software. ( K ) Immunoblot analysis of JEV NS5 SUMOylation in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, ZNF33B-MYC, and the WT or mutant construct of NS5-FLAG, with arginine replacement of the SIM (V-I-D-L to 4R). The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( L ) Immunoblot analysis of the SIM mutations on the association of ZNF33B with JEV NS5 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-MYC and NS5-FLAG. The cell lysates were immunoprecipitated with anti-FLAG antibody.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Western Blot, Transfection, Mutagenesis, Construct, Immunoprecipitation, Microscopy, Sequencing, Derivative Assay, Generated, Software

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: SUMOylation of JEV NS5 hinders its ubiquitination through competitive binding to lysine residues 269 and 846. ( A ) Immunoblot analysis of the effect of JEV NS5 SUMOylation on its ubiquitination in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, NS5-FLAG, and Ubi-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( B ) Immunoblot analysis of the effect of JEV NS5 deSUMOylation on its ubiquitination in HEK293T cells co-transfected with NS5-FLAG and Ubi-HA, followed by 2-D08 (200 µM) treatment for 6 h. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( C–E ) Immunoblot analysis of the K269R ( C ), K287R ( D ), and K846R ( E ) mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant constructs of NS5-FLAG, with arginine replacement of the lysine residues at positions 269 ( C ), 287 ( D ), and 846 ( E ). The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F ) Immunoblot analysis of the SIM mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant construct of NS5-FLAG, with arginine replacement of the SIM (V-I-D-L to 4R). The cell lysates were immunoprecipitated with anti-FLAG antibody.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Transfection, Immunoprecipitation, Mutagenesis, Construct

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: ZNF33B recruits HSPB1 to facilitate JEV NS5 SUMOylation. ( A ) Silver staining of JEV NS5-associated proteins immunoprecipitated by anti-HA antibodies probe with cell lysates from SK6 cells. The arrows indicate the additional band presented in JEV NS5-associated proteins. ( B and C ) Differentially expressed proteins analyzed by mass spectrometry. ( D and E ) Immunoblot analysis of the association of NS5 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with NS5-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F and G ) Confocal microscope observation of the colocalization of NS5 with HSPB1 and HSPB8 in HEK293T cells transfected with NS5-FLAG with HSPB1-HA or HSPB8-HA. Scale bar, 2 µm. ( H and I ) Immunoblot analysis of the expression of JEV NS3 and NS5 in HEK293T cells transfected with HSPB1-HA or HSPB8-HA, followed by JEV infection. ( J and K ) Immunoblot analysis of the effect of HSPB1 and HSPB8 on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and HSPB1-HA or HSPB8-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( L ) Immunoblot analysis of the association of UBC9 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with UBC9-V5 and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( M ) Immunoblot analysis of the association of ZNF33B with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( N ) Immunoblot analysis of HSPB1 and HSPB8 in HEK293T cells transfected with ZNF33B-HA, followed by JEV infection. The expression of HSPB1 and HSPB8 was assessed by measuring the band grayscale with the “ImageJ” software. ( O ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on the protein level of ZNF33B-promoted NS5 in HEK293T cells transfected with ZNF33B-MYC, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( P ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on ZNF33B-promoted JEV NS5 SUMOylation from HEK293T cells co-transfected with ZNF33B-MYC, pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Silver Staining, Immunoprecipitation, Mass Spectrometry, Western Blot, Transfection, Microscopy, Expressing, Infection, Software