Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation intricately regulates increased expression in CAFs. ( A ) SUMOylation sites of FOXL2 was analyzed by SUMOplot™ analysis. Shown are the top 7 predicted lysine residues; ( B ) In vitro sumoylation assay was employed to confirm the predicted SUMOylation sites using HA-tagged wild-type (WT), or the K25R, K87R, K114R, K150R, and 4KR (where K25, K87, K114 and K150 were all mutated to R). Shown is a representative blot and densitometry analysis of SUMOylated-FOXL2/Total FOXL2; ( C ) The association between SUMOylation and FOXL2 stability was determined by western blotting in HEK-293T cells using α-HA. Cells were transfected with either HA-FOXL2-WT or HA-FOXL2-K25/87R (double mutant, 2KR). The membrane was stripped and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( D ) Cells were transfected with HA-FOXL2-WT or, HA-FOXL2-2KR ± His-SUMO1. CHX (100 µg/ml) was added to inhibit translation allowing tracking of FOXL2 stability in the presence and absence of His-SUMO1. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( E ) DMSO (control), MG132 (proteasome inhibitor) or chloroquine (lysosome inhibitor) were added into HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, before CHX (100 µg/ml) was added. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot; ( F ) IP assay was used to test the association between FOXL2 SUMOylation and ubiquitination. Lysates obtained from HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, along with FLAG-UBC9, were immunoprecipitated using α-HA antibody and then probed with α-FLAG antibody. Shown is a representative blot; *, *** P < 0.05, P < 0.001

Article Snippet: The different primary antibodies used were α-SUMO1 (67559-1-Ig, 1:3000), α-UBC9 (10070-1-AP, 1:2000), α-Vimentin (10366-1-AP, 1:5000), α-N-cadherin (22018-1-AP, 1:5000), α-E-cadherin (20874-1-AP, 1:20000) (Proteintech, Wuhan, China); α-HA (A02041, 1:800), α-His (A02050, 1:800), α-Flag (A02010, 1:800) (Abbkine, Wuhan, China); α-FOXL2 (ab246511, 1:1000), α-Snail (ab216347, 1:1000) (Abcam, Cambridge, MA, USA); and, α-Smad2/3 (D7G7, 1:1000), α-p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, 1:1000) (CST, Boston, MA, USA).

Techniques: Expressing, In Vitro, Western Blot, Transfection, Mutagenesis, Membrane, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation requires SUMO1 and UBC9/UBE2I. ( A ) Putative interaction of FOXL2 with SUMO1, SUMO2, SUMO3, and SUMO4 was detected by String analysis ( https://cn.string-db.org/ ); ( B ) In vitro sumoylation assay was employed to confirm String analysis’s prediction of SUMO1, HEK-293T cells were transfected with HA-FOXL2-WT, FLAG-UBC9, and His-SUMO1-4. In vitro SUMOylation assay using Ni 2+ -NTA pull-down determined that FOXL2 was mainly modified by SUMO1. Shown is a representative blot; ( C ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation. HEK-293T cells were transfected with HA-FOXL2-WT and His-SUMO1 ± FLAG-UBC9. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot; ( D ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation in CAFs. CAFs were transduced using either a non-targeting control shRNA or shRNA targeting UBC9 . Transduced cells were transfected with HA-FOXL2-WT and His-SUMO1. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot

Article Snippet: The different primary antibodies used were α-SUMO1 (67559-1-Ig, 1:3000), α-UBC9 (10070-1-AP, 1:2000), α-Vimentin (10366-1-AP, 1:5000), α-N-cadherin (22018-1-AP, 1:5000), α-E-cadherin (20874-1-AP, 1:20000) (Proteintech, Wuhan, China); α-HA (A02041, 1:800), α-His (A02050, 1:800), α-Flag (A02010, 1:800) (Abbkine, Wuhan, China); α-FOXL2 (ab246511, 1:1000), α-Snail (ab216347, 1:1000) (Abcam, Cambridge, MA, USA); and, α-Smad2/3 (D7G7, 1:1000), α-p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, 1:1000) (CST, Boston, MA, USA).

Techniques: In Vitro, Transfection, Modification, Pull Down Assay, Control, shRNA

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells with or without STM 14028S infection (4 hpi). Scale bar, 10 µm. b Core SUMO cycle enzymes and their corresponding primary genes (blue). c, d Transcriptomic and proteomic analyses of SUMO cycle enzyme expression in RAW264.7 cells infected with STM 14028S vs uninfected controls (4 hpi; n = 3). e UBC9 mRNA expression in RAW264.7 cells during STM 14028S infection (0-6 hpi; n = 3). f Immunoblot analysis and quantification of UBC9 protein levels in STM 14028S-infected RAW264.7 cells (0-6 hpi; n = 3). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, Expressing, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM 14028S over a 0-6 hrs time course. Scale bar, 10 µm. b Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM WT or Δ ssaV or left uninfected (4 hpi). Scale bar, 10 µm. c Schematic of the high-content screening (HCS) workflow used to identify T3SS-2 effector(s) required for subversion of host SUMOylation. d SUMO2/3 suppression rates in RAW264.7 cells infected with STM WT or T3SS-2 effector knockout strains (4 hpi). For each sample, fluorescence intensity (FI) was measured across three fields (20 cells per field) to calculate the mean FI (MFI). Suppression rate = (MFI_uninfected - FI_test) / (MFI_uninfected - MFI_WT). e Representative immunofluorescence images of SUMO2/3 conjugation in RAW264.7 cells infected with STM WT, Δ ssaV , Δ sseK1 , or left uninfected (4 hpi). f Immunoblot analysis of SUMO2/3 conjugates in RAW264.7 cells infected with STM WT or Δ sseK1 or left uninfected (4 hpi). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, High Content Screening, Knock-Out, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK2, or SseK3. b AlphaFold-predicted structures of SseK1 (aa 29-336, red), SseK2 (aa 29-348, yellow), and SseK3 (aa 29-335, green), shown with structural alignment using PyMOL. c AlphaFold-modeled structure of the SseK1-UBC9 complex. The enlarged view of the lid-domain region is boxed in black. Residues in the SseK1 lid domain forming hydrogen bonds with UBC9 are shown in stick representation. d Sequence alignment of SseK1, SseK2, and SseK3 generated using ESPript 3.0. e Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK1 A332_Q336del, SseK3, and SseK3 R332delinsARHVQ. f Immunoblot analysis of SUMO2/3 conjugation in RAW264.7 cells infected with STM Δ sseK1 , STM Δ sseK1 complemented with SseK1, or STM Δ sseK1 complemented with either SseK1 D223_D225delinsAAA or SseK1 A332_Q336del (4 hpi). g Phylogenetic analysis of Salmonella Typhimurium SseK1 homologs. Protein sequences homologous to SseK1 (UniProt accession: A0A0H3NK84) were identified using BLASTP analysis against the UniProtKB reference proteomes and Swiss-Prot databases. Sequences with an E-value < 0.05 were selected for phylogenetic analysis. The phylogenetic tree was constructed and visualized using the Interactive Tree of Life (iTOL) online tool.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Western Blot, Sequencing, Generated, Conjugation Assay, Infection, Construct

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Protein SUMOylation sites showing a >3-fold increase in SUMOylation intensity in STM Δ sseK1 -infected cells relative to WT. SUMOylation intensity was normalized to the corresponding protein abundance measured in proteomic datasets from STM Δ sseK1 - or WT-infected RAW264.7 cells. b GO and KEGG pathway enrichment analysis of proteins with SUMOylation intensity ratio (Δ sseK1 /WT) > 3. c, d IP analysis of Myd88 and Hspa8 SUMO2/3 modification in RAW264.7 cells infected with STM WT or Δ sseK1 or left uninfected (4 hpi). e Immunoblot analysis of SUMO2/3 modification in RAW-SseK1-OE and RAW-GFP-Ctrl cells. f Nine-quadrant plot showing protein abundance changes in two proteomic comparisons (RAW-SseK1-OE vs RAW-GFP-Ctrl and STM WT-infected vs Δ sseK1 -infected cells). Highlighted proteins are downregulated in both datasets. Fold-change values within dashed lines indicate <|1.3|. g Immunoblot analysis of PDCD4 expression in RAW-SseK1-OE and RAW-GFP-Ctrl cells. h Immunoblot analysis of PDCD4 and SUMO2/3 levels in RAW264.7 cells treated with increasing concentrations of the SUMOylation inhibitor 2-D08. ***P < 0.001.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Infection, Quantitative Proteomics, Modification, Western Blot, Expressing

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Intracellular survival of STM WT and Δ sseK1 strains in RAW264.7 cells (1-6 hpi). n = 3. b Schematic of the experimental workflow for 2-D08 treatment and Salmonella infection of RAW264.7 cells. c Intracellular survival of STM WT and Δ sseK1 strains in RAW264.7 cells with or without 2-D08 pretreatment (4 hpi). d Schematic of the experimental workflow for 2-D08 administration and STM challenge in C57BL/6 mice. e Immunoblot analysis of SUMO2/3 modification in liver, spleen, and intestine tissues from C57BL/6 mice challenged with STM WT or Δ sseK1 , with or without 2-D08 pretreatment. f Bacterial load (CFU per gram of liver or spleen) in C57BL/6 mice infected with STM WT or Δ sseK1 . n = 4. g Survival curves of C57BL/6 mice challenged with STM WT or Δ sseK1 , with or without 2-D08 pretreatment. n = 6. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Infection, Western Blot, Modification

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Depletion of Proteostasis Factors by Chronic HSF1 Inhibition Delays Recovery to Normal SUMO2/3 Conjugation Levels after Heat Shock (A) Use of a dominant-negative, constitutively active HSF1 construct (dn-cHSF1), created via disruption of the regulatory domain (RD) by removing amino acids 186–202 combined with deletion of the transcription activation domain (TAD), for inhibition of endogenous HSF1. (B) Western blot analysis of HSP40 protein levels in response to different time periods of Dox-induced dn-cHSF1 induction in HEK293T-REx cells. (C) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS). Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP40 levels were used to confirm the induction of the heat shock response and functional HSF1 inhibition. (D) HEK293T-REx cells constitutively expressing DHFR.dn-cHSF1 were treated with trimethoprim (TMP) for 48 hr (chronic HSF1 inhibition) or 4 hr (acute HSF1 inhibition) prior to HS. Cells were subsequently exposed to HS and recovery as described in (A). Total amounts of SUMOylated proteins were determined by immunoblotting. (E) Quantification of total SUMO2/3 conjugation from (D) as compared to SUMOylation post-heat shock in basal proteostasis conditions. n = 3, error bars indicate SD. Significance was determined using ANOVA analysis followed by post hoc Tukey analysis, ∗ p < 0.05; ∗∗ p < 0.005, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Conjugation Assay, Dominant Negative Mutation, Construct, Activation Assay, Western Blot, Expressing, Functional Assay

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Proteomic Identification of SUMOylated Proteins Whose Recovery to Normal SUMO-Conjugation Levels Post Heat Shock Is Delayed by Chronic HSF1 Inhibition (A) Workflow for Figure 2 . HEK293T-REx cells stably co-expressing His10-SUMO2 and either Dox-inducible dn-cHSF1 or Dox-inducible GFP were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS). Cells were exposed to HS at 43°C for 75 min before returning to 37°C for a recovery period (rec). Red arrows indicate time points at which cells were lysed. SUMOylated proteins were purified by means of His10 pull-down, proteins were trypsinized, and peptides were analyzed by mass spectrometry. (B) Immunoblotting of cell lysates from (A) using antibodies against SUMO2/3, HSP70, and GFP. Total amounts of proteins in each lane were visualized by Ponceau S staining. (C) Volcano plots depicting statistical differences in abundance between proteins identified by mass spectrometry. Dashed lines indicate a cut-off at a p value ≤ 0.05 (–log10 p value ≤ 1.3) and a fold change of 2 (log2 = 1). The left panel shows SUMOylated proteins identified immediately post-HS in the Dox-inducible dn-cHSF1 cell line, specifically in the Dox-treated sample compared to the untreated sample. The right panel shows SUMOylated proteins identified 4-hr post-HS in the Dox-inducible dn-cHSF1 cell line compared to the untreated sample. Proteins marked in red are either HSF1 or proteins that were further validated. (D) Selection of enriched gene ontology terms of biological processes and molecular functions for selected proteins that significantly retained SUMOylation 4-hr post-HS when HSF1 activity was chronically inhibited. See also and and , , , and .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Conjugation Assay, Inhibition, Stable Transfection, Expressing, Purification, Mass Spectrometry, Western Blot, Staining, Selection, Activity Assay

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Chronic HSF1 Inhibition Interferes with Degradation of SUMOylated and Ubiquitinated Proteins during Heat Shock Recovery (A) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i), with 100 nM bortezomib (proteasome inhibition) immediately prior to heat shock (HS), or with a combination of both. Cells were exposed to HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP40 levels were used to confirm induction of the heat shock response and functional HSF1 inhibition. (B) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were treated as in (A). Total amounts of ubiquitinated proteins were analyzed by immunoblotting. (C) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated as in (A). SUMOylated proteins were purified by means of His10 purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (D) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A). Ubiquitinated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. See also and .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Expressing, Western Blot, Functional Assay, Stable Transfection, Purification, Staining

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: Chronic HSF1 Inhibition Selectively Impairs Degradation of SUMO2/3 and Ub Co-modified Proteins during Heat Shock Recovery (A) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS), with 100 nM bortezomib (proteasome inhibition) immediately prior to HS, or with a combination of both. Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Elutions were analyzed by immunoblotting for Ub and SUMO2/3. (B) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A). Elutions were analyzed by immunoblotting for SUMO2/3 and Ub. (C) Quantification of (B). Graph shows fold change of the ratio of SUMOylated proteins over ubiquitinated proteins in His10-Ub purified elutions as compared to the ratio after HS under basal proteostasis conditions. n = 3, error bars represent SD. Significance was determined using ANOVA followed by post hoc Tukey’s multiple comparison test, ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Inhibition, Modification, Stable Transfection, Expressing, Western Blot, Purification

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: HSP90 Plays a Key Role in the Recovery of SUMO2/3 Modification following Heat Shock (A) HEK293T-REx cells expressing Dox-inducible dn-cHSF1 were either treated with Dox for 48 hr (chronic HSF1i) prior to heat shock (HS), with 500 nM STA-9090 (HSP90i) 2 hr prior to HS, or with a combination of both. Cells were exposed to a HS at 43°C for 75 min before returning to 37°C for a recovery period (rec) and lysed as indicated. Total amounts of SUMOylated proteins were analyzed by immunoblotting. HSP70 levels were used to confirm induction of the heat shock response and functional HSF1 inhibition. (B) Quantification of fold change of SUMOylation from (A) as compared to SUMOylation after HS under the basal proteostasis conditions. n = 3, error bars indicate SD. Significance was determined using ANOVA analysis followed by post hoc Tukey analysis, ∗ p < 0.05; ∗∗∗ p < 0.0005. (C) HEK293T-REx cells stably co-expressing His10-SUMO2 and Dox-inducible dn-cHSF1 were treated as in (A) with a 2-hr recovery at 37°C. SUMOylated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (D) As for (C). Elutions were analyzed by immunoblotting for Ub and SUMO2/3. (E) HEK293T-REx cells stably co-expressing His10-Ub and Dox-inducible dn-cHSF1 were treated as in (A) with a 2-hr recovery at 37°C. Ubiquitinated proteins were purified by means of His10-purification. Elutions and inputs were analyzed by immunoblotting for FoxM1. Ponceau S stain was used as a loading control for inputs. (F) As for (E). Elutions were analyzed by immunoblotting for SUMO2/3 and Ub. See also Figure S6 .

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Modification, Expressing, Western Blot, Functional Assay, Inhibition, Stable Transfection, Purification, Staining

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet: SUMO2/3 Plays a Key Role as an Integrated Component of the Proteotoxic Stress Response Upon HS, a transient increase in SUMO2/3 conjugation to target proteins is observed, which may function in part to prevent irreversible aggregation of target proteins (reduced aggregation was demonstrated in vitro ). SUMO2/3-modified proteins are frequently co-modified by Ub, and a large fraction of these co-modified proteins are subsequently degraded by the proteasome during HS recovery in a chaperone-dependent manner.

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Conjugation Assay, In Vitro, Modification

Journal: Cell Reports

Article Title: SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock

doi: 10.1016/j.celrep.2018.12.027

Figure Lengend Snippet:

Article Snippet: His 10 -SUMO2-WT-IRES-GFP expressing cells were sorted by fluorescence-aided cell sorting (FACS) on the BD FACSAriaIII™ cell sorter for low GFP expression to limit His 10 -SUMO2 expression.

Techniques: Recombinant, SYBR Green Assay, Plasmid Preparation, Software