Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: (A) Schematic showing the design of dual-gRNA directed CRISPR screen of E2s regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format. (B) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937-Cas9_ePL-IKZF1 parental cells or cells expressing UBE2G1-specfic sgRNA alone or in combination with non-targeting or UBE2D3-specific sgRNA. Cells were treated with POM at the indicated concentrations for 16 hours. Data are presented as mean ± SD (n = 4). (C) Immunoblot analysis of U937-Cas9 parental cells or cells expressing non-targeting sgRNA, UBE2G1-specific sgRNA, UBE2D3-specfic sgRNA, or both UBE2G1 and UBE2D3 sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hours. SE, short exposure; LE, long exposure.

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: CRISPR, Expressing, Western Blot

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2G1 catalyzes the ubiquitin chain assembly on GSPT1 pre-conjugated with ubiquitin (A) Sequence alignment of human UBE2G1, human UBE2G2 and human CDC34 using Clustal W 2.1. The acidic loops indispensable for the assembly of K48-linked ubiquitin chains are highlighted with red, and the catalytic cysteines are highlighted with blue. (B) Immunoblot analysis of 293T parental or UBE2G1-/- cells transduced with lentiviral vectors expressing FLAG-tagged UBE2G1 wild-type or C90S mutant, or FLAG-tagged UBE2D3 wild-type or C85S mutant. Cells were treated with CC-885 at the indicated concentrations for 4 hours. Note that overexpression of wild-type FLAG-UBE2G1 or FLAG-UBE2D3 partially rescued the GSPT1 degradation defect caused by UBE2G1 deficiency, while overexpression of catalytically-dead mutant FLAG-UBE2G1-C90S or FLAG-UBE2D3-C85S further blocked the degradation of GSPT1. (C) In vitro ubiquitination of GSPT1 by CRL4 CRBN with or without CC-885 and indicated E2 variants. Consistent with results observed with bacterial recombinant UBE2G1 and UBE2D3 proteins, FLAG-UBE2G1 and FLAG-UBE2D3 proteins purified from human cells acted in concert to promote the ubiquitination of GSPT1.

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Sequencing, Western Blot, Transduction, Expressing, Mutagenesis, Over Expression, In Vitro, Recombinant, Purification

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: (A-D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion proteins by recombinant CRL4CRBN complex. Recombinant protein products as indicated were incubated with or without 80 μM POM (A and C) or 80 μM CC-885 (B and D) in the ubiquitination assay buffer containing 80 mM ATP at 30°C for 2 hours, and then analyzed by immunoblotting. (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN complex. MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1, DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30 °C for 4 hours. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30 °C for 2 hours, followed by immunoblot analysis. (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1.

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot, Purification, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: (A and B) 293T parental and UBE2G1-/-;UBE2D3-/- (clone 4) cells were transiently transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or without UBE2G1, UBE2D3 or both. (C) 293T parental and UBE2G1-/- (clone 13) cells were transiently transfect with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hours after transfection, cells were treated with MG-132 (10 μM) and POM at the indicated concentrations for additional 8 hours. Ubiquitinated protein products enriched with magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis of whole cell extracts showing equal input proteins is shown in .

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: Input protein levels for the in vivo ubiquitinaiton studies corresponding to (A) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/- ; UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, cereblon and IKZF1-V5. (B) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/-;UBE2D3-/- (Clone 4) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 and/or UBE2D3. (C) Total input for . Immunoblot analysis of 293T parental and UBE2G1-/- (Clone 13) cells transfected to produce 8xHis-Ubiquitin, CRBN, IKZF1-V5 with or without UBE2G1 wildtype or C90S mutant. In (A), (B) or (C), 48 hours after transfection, cells were treated with 10 μM MG132 and POM at the indicated concentrations for additional 8 hours.

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: In Vivo, Western Blot, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

doi: 10.1101/389098

Figure Lengend Snippet: UBE2D family proteins redundantly promote the ubiquitination of GSPT1 (A) Sequence alignment of human UBE2D family proteins using Clustal W 2.1. Note that the amino acid sequence identity among all 4 family proteins is close to 90%. (B) In vitro ubiquitination of GSPT1 MBP fusion protein by recombinant CRL4CRBN complex in the presence of UBE2G1, UBE2D1, UBE2D2, or UBE2D3, alone or in combination. Recombinant protein products as indicated were incubated with or without 80 μM CC-885 in the ubiquitination assay buffer at 30 °C for 2 hours, and then analyzed by immunoblotting. SE, short exposure; LE, long exposure

Article Snippet: Purified recombinant human Ube1 E1 (E-305), UbcH5a/UBE2D1 (E2-616-100), UbcH5b/UBE2D2 (E2-622-100), UbcH5c/UBE2D3 (E2-627-100), wild-type ubiquitin (U-100H), K48R ubiquitin (UM-K48R-01M), and K48-only ubiquitin (UM-K480-01M) were purchased from R&D systems.

Techniques: Sequencing, In Vitro, Recombinant, Incubation, Ubiquitin Assay, Western Blot

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Schematic representation of the recruitment of PRC2.1 and PRC2.2. MTF2 binds to DNA, while the EED subunit of core PRC2 (orange) binds to H3K27me3 as part of an allosteric feedback loop. The EZH2 subunit of core PRC2 catalyses H3K27 methylation. The PRC2.2 complex contains JARID2 but not MTF2. Both contain the core PRC2 subunits, however the interactions of the PRC2.1 and PRC2.2-specific subunits with chromatin are different. The arrow from JARID2 to DNA is dashed as DNA binding has been shown in vitro but not in vivo b) PRC2.1 (MTF2) and PRC2.2 (JARID2) co-localize to all EZH2 targets. c-f) Heatmap and rpkm quantification (boxplots) of PRC2 subunits and the catalytic product H3K27me3. EZH2 recruitment is heavily affected by the absence of MTF2, while JARID2 and H3K27me3 absence have minor effects (c). The effect of MTF2 and JARID2 on EZH2 recruitment is reflected on H3K27me3 deposition (d). MTF2 is marginally affected by H3K27me3 removal, but its binding is reduced to approximately half the WT level in the absence of JARID2 (e). JARID2 recruitment is strongly reduced in the absence of either H3K27me3 or MTF2 (f). ChIP profiles are highly reproducible g) Genome browser examples of PRC2 binding to classical Polycomb targets. Box plots represent median and interquartile range (IQR; whiskers, 1.5 IQR).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Methylation, Binding Assay, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a-d) Heatmap of WT ChIP-seq signal on the indicated peak set. H3K27me3-negative JARID2 peaks were excluded from further analysis. e) Venn diagram showing the overlap of peaks called for the ChIP-Seq of each protein independently. f) Mass spectrometry quantification of PRC2 subunits in the different cell lines. Detection of JARID2 and MTF2 in the respective mutants (asterisks) is due to value imputation in Perseus. g) Western blot validation of EED226 depletion of H3K27me3, for the ChIP shown in Figs 1 and 2. h) Scatterplot of peak RPKM showing high reproducibility of ChIP replicates.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: ChIP-sequencing, Mass Spectrometry, Western Blot

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Clustering of all PRC2 targets using ChIPseq data in multiple PRC2 mutants. Cluster 1-4 are unmethylated CpG islands (strong BioCap) signal, showing bivalent marks in WT (H3K4me3 and H3K27me3). These regions display heavy reduction of EZH2 recruitment in the MTF2 mutant, milder effects of H3K27me3 absence (EED226 treatment), and little or no effect of JARID2 absence. The intensity of MTF2 binding depends on both H3K27me3 and JARID2 but binding is still clearly detectable even in the absence of PRC2 core ( Eed -/- ) indicating a primary binding to DNA, reinforced by other mechanisms, such as JARID2-mediated recruitment, which in turn also depends on both H3K27me3 and MTF2. Cluster 5 and 6 have lower BioCap and H3K4me3 signal, and, while still affected by the absence of MTF2, this has a much less marked effect on recruitment of both EZH2 and JARID2, and on H3K27me3 deposition. b) WT-normalized, input-subtracted RPKM quantification of signal shown in (a). c) Quantification of GCG trinucleotides matching DNA shape requirement for MTF recruitments as defined in . Cluster 1-4 are strongly enriched in shape-matching GCGs, indicating potential for strong DNA-mediated MTF2 recruitment. d) Enrichment of anatomical terms in the genes associated with peaks in the six clusters. Enrichment within PRC2 targets. Cluster 4 show strong enrichment for CNS structures, cluster 5 and 6 for limb and branchial arches tissues and mesenchyme. See for the full overview.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing the cluster specific effect of H3K27me3 depletion on the binding of EZH2. WT and MTF2 GT/GT show mild reduction of EZH2 binding when treated with EED226 inhibitor, while the treatment is highly synergistic with the depletion of JARID2. b) Bootstrapping-based RPKM quantification (methods) of the signal in (a). Each coloured dot represent the median of one round of bootstrapping, grey bar represent 99.9% confidence interval for the mean of bootstrapped values in each condition and cluster. c) Treatment with EED226 further affected MTF2 recruitment in Jarid2 -/- and JARID2 recruitment in Mtf2 GT/GT , with the former leading to recruitment patter closely resembling the Eed -/- line (cf. ), highlighting the recruitment differences between cluster 1-4 and 5-6. d) Bootstrapping-based RPKM quantification (methods) of the signal in (c) similar as in 3b. e) Genome browser view of example Polycomb targets. For each genotype two tracks are overlaid: the darker colour represent EED226 treated samples, the lighter colour untreated cells.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing EZH2, MTF2 and JARID2 binding in the absence of H3K27me3 in PRC2 and PRC1 mutant lines. In the absence of H3K27me3, JARID2 and RING1A/B mutant phenocopy each other with regard to EZH2 and MTF2 binding, suggesting JARID2 and RING1B act in the same PRC2 recruitment mechanism. JARID2 recruitment is also strongly affected by the absence of RING1A/B, in line with the JARID2-mediated PRC2 recruitment via binding to PRC1-deposited H2AK119ub. b-d) Average plot of the ChIP signal shown in (a), for EZH2 (b) MTF2 (c) and JARID2 (d) centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization. e) Heatmap showing Ring1b binding in the discussed conditions. Ring1b is only mildly affected by removing H3K27me3 using EED226 (~40%). Binding is further attenuated in MTF2 and JARID2 mutant ESCs. f) Average plot of the ChIP signal shown in (e), centred on called peaks. g) Examples of loci of the data as shown in (e).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) On PRC2.1 main targets (clusters 1-4) relatively little MTF2 binding is sufficient to kick start the EED positive feedback loop which heavily relies on JARID2. As primary recruitment is mediated to a large extent via MTF2, such a loop can still exist in the absence JARID2. In the absence of H3K27me3, an alternative route can take over that requires JARID2 binding to H2AK119ub. b) On PRC2.2/PRC1 targets (clusters 5-6), instead, Polycomb binding is initiated by PRC1 that, upon H2AK119ub deposition, is followed by JARID2-containing PRC2.2. These regions also see the presence of MTF2 in physiological conditions, but this is the result of indirect recruitment via the PRC2 core binding to PRC2.2-initiated H3K27me3 deposition.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: ErbB2, septin-9, septin-2, and septin-7 expression were determined by western blot analysis of total lysates of various cell lines and human gastric antrum, β-actin was used as a control. Cofilin was used as a control for septin-2 given the size similarity between septin-2 and β-actin. ErbB2 is very highly expressed in HGE-20 gastric cancer cells, to the extent that the band is not visible in other cell types with reasonable exposure of the HGE-20 cell band. Once HGE-20 cells were removed from the ErbB2 blot, expression was also seen in human gastric antrum, HB2 breast cancer cells, and AGS gastric cancer cells. Septin-9 similarly showed increased expression in gastric cancer cells compared to other cell types.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Expressing, Western Blot, Control

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: Proteins immunoprecipitated using antibodies against septin-2 or ErbB2 from HGE-20 cell lysates were analyzed by mass spectrometry. ErbB2, known ErbB2-associated proteins, were detected in septin-2 immunoprecipitate and septin-9 was detected in ErbB2 immunoprecipitate (A). Interaction of ErbB2 with septins and a known dimerization partner, EGFR, was confirmed by western blot, following immunoprecipitation of ErbB2 in HGE-20 cell lysate. Ubiquitin ligase c-cbl binding protein, CIN85, a protein that mediates the interaction between septin-9 and EGFR, was not seen in ErbB2 IP (B). Interaction between septin-9 and ErbB2 was again confirmed by western blot following IP of HGE-20 cell lysates with septin-9 antibody (C). emPAI-exponentially modified protein abundance index, UR-unrelated antibody negative control (anti-human influenza hemagglutinin for ErbB2 IP and anti-green fluorescent protein for septin-9 IP).

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot, Ubiquitin Proteomics, Binding Assay, Modification, Quantitative Proteomics, Negative Control

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: ErbB2 protein is localized mainly to the basolateral membrane of HGE-20 cells as shown by confocal microscopy (A). ErbB2 and septin-2 co-localize at the lateral membrane in HGE-20 cells, as shown by immunofluorescence using antibodies against ErbB2 and septin-2 (B).

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Membrane, Confocal Microscopy, Immunofluorescence

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: HGE-20 cells were incubated with FCF or vehicle for 12 hours, followed by fixation and immunofluorescence. The images show an altered distribution of septin-2 (A) and septin-9 (B) and a decrease in co-localization between septins and ErbB2 (A-B) in the presence of FCF. Coefficients of co-localization as calculated using Zen 2009 software are shown below the images. Errors bars represent s.d., n=3 independent experiments, at least 12 fields were analyzed for each condition; *-significant difference from no FCF control, P <0.05, Student’s t-test.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Incubation, Immunofluorescence, Software, Control

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: AGS cells were transiently transfected with septin-2-specific or control (ctr) siRNA or AGS and HGE-20 cells were incubated with FCF for 12 hours. Western blot was performed on clarified lysates. ErbB2 protein levels were decreased with siRNA silencing of septin-2. Use of FCF led to a similar decrease in ErbB2 protein in both cell lines. The structurally similar EGFR was not affected, demonstrating specificity of response. β-actin was used as a loading control (A). Immunofluroescence with ErbB2 antibodies in HGE-20 cells showed accumulation of ErbB2 in intracellular compartments following 6 hour incubation with FCF, with overall decreased expression and minimal intracellular appearance of ErbB2 at 12 hours, suggestive of intracellular degradation (B). Arrows in the images point to intracellular accumulation of ErbB2. Numbers below images indicate the intensity of total fluorescence as % of control as calculated by analyzing at least 10 microscopic fields for each condition using Zen 2009 software. n=3 independent experiments, errors or error bars represent standard deviation (s.d.), *-significant difference from condition without siRNA or FCF, p<0.05, Student’s t-test.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Transfection, Control, Incubation, Western Blot, Expressing, Fluorescence, Software, Standard Deviation

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: HGE-20 cells were incubated with FCF for 12 hours, followed by immunoprecipitation with antibodies against septin proteins. ErbB2 was decreased in septin immunoprecipitates and cell lysates in the presence of FCF, while septin protein levels were unchanged. IgG refers to immunoprecipitation procedure without cell lysate (A). HGE-20 cells were incubated with or without FCF for 12 hours and lysed. The level of septin-9 was depleted in HGE-20 cell lysates by 4 consecutive immunoprecipitations with septin-9 antibodies, each using the supernatanat of the prior preparation. The level of septin-9 was clearly depleted by the 4th immunoprecipitation (B). Western blot analysis of the final supernatant from (B) detects more ErbB2 in a sample from FCF-treated cells as compared to control. Western blot analysis of ErbB2 immunoprecipitates from the final supernatant from (B) shows less septin-2 and septin-9 bound to ErbB2 in the sample from FCF-treated cells (C). n=3 independent experiments, error bars represent s.d., *-significant difference from no FCF condition, P<0.05, Student’s t-test.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Incubation, Immunoprecipitation, Western Blot, Control

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: There was no difference in FCF-induced decrease in ErbB2 in the presence or absence of cycloheximide, as shown by western blot of total HGE-20 cell lysate, suggesting the effect of FCF is on mature ErbB2 protein (A). Incubation with FCF or vehicle was completed for 12 hours with or without cycloheximide, followed by immunoprecipitation with ErbB2 antibodies. Western blot using ubiquitin antibodies, followed ErbB2 antibodies, showed increased ubiquitylation of ErbB2 in the presence of FCF, independent of new protein synthesis (B). Ubiquitylation induced by FCF is specific to ErbB2, as shown by western blot comparison of ErbB2 immunoprecipitate and total cell lysate using ubiquitin antibodies; β-actin was used as a loading control (C). Cells were incubated with FCF or vehicle, surface proteins were biotinylated from basolateral side, followed by immunoprecipitation with ErbB2 antibodies (IP1), then boiling of eluted proteins in SDS to remove interacting proteins, followed by dilution in non-ionic detergent and repeat immunoprecipitation with ErbB2 antibodies (IP2). Loss of septin-9 and cofilin signals on western blot in IP2 confirms dissociation of ErbB2 from interacting proteins. Increased ubiquitylation of ErbB2 is still seen in the absence of interacting proteins, demonstrating that ErbB2 itself is ubiquitylated. Proteins eluted from the second IP by boiling in SDS were diluted in non-ionic detergent and biotinylated ErbB2 was isolated by streptavidin extraction (SA). As shown by western blot with ubiquitin antibodies, the increased ubiquitylation of ErbB2 induced by FCF is seen in this fraction, demonstrating ubiquitylation occurs at the plasma membrane. The graph shows the FCF-increased ubiquitylation seen in each experiment as compared to control (D). Ub-ubiquitylated ErbB2, UM-unmodified ErbB2, IgG-immunoprecipitation control without cell lysate, SA-streptavidin.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Western Blot, Incubation, Immunoprecipitation, Ubiquitin Proteomics, Comparison, Control, Isolation, Extraction, Clinical Proteomics, Membrane

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: HGE-20 cells were incubated with the indicated inhibitors for 6 hours, followed by basolateral biotinylation of surface proteins, streptavidin extraction, and western blot using anti-ErbB2 antibodies. Na+,K+-ATPase β1 subunit was used as a loading control. ErbB2 protein levels in the membrane fraction were not protected by any of the indicated inhibitors in the presence of FCF (A). In total cell lysates, ubiquitylated ErbB2, seen as an increased density running above the main ErbB2 band (arrowhead), was increased in the presence of cathepsin B inhibitor CA074-me (B). ErbB2 was immunoprecipitated, followed by western blot using anti-ubiquitin, then anti-ErbB2 antibodies, confirming a significant increase in ubiquitylated forms of ErbB2 in the presence of CA074-me and also showing a moderate increase in the presence of lactacystin (C). Quantification for each blot is shown to the right. Error bars, s.d., n=4 independent experiments, statistics done by Student’s t-test, ns-not significant, * - significant difference from the no-FCF control, P<0.05, ** - significant difference between indicated conditions, p<0.05, CA-CA074-me, L-lactacystin, B-bafilomycin, V-vehicle, Ub-ubiquitylated form of ErbB2, UM-unmodified form of ErbB2.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Incubation, Extraction, Western Blot, Control, Membrane, Immunoprecipitation, Ubiquitin Proteomics

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: HGE-20 cells were incubated with the indicated inhibitors for 6 hours, then fixed. Immunofluorescence was performed using antibodies against ErbB2 and the lysosomal protein LAMP2. There was an increase in co-localization of ErbB2 with lysosomes in the presence of FCF and cathepsin B inhibitor CA074-me. Coefficient of overlap between ErbB2 and LAMP2 as calculated using Zen 2009 software with the confocal microscope is shown below the images (A). Low magnification images of HGE-20 cells incubated with FCF and CA074-me show that lysosomal accumulation of ErbB2 occurs in the majority of cells in a cell monolayer (B). Error bars, s.d., n=3 independent experiments, at least 12 fields were analyzed for each condition, * - significant difference from no-FCF condition, P = 0.0007, Student’s t-test.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Incubation, Immunofluorescence, Software, Microscopy

Journal: The Biochemical journal

Article Title: Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

doi: 10.1042/BCJ20160203

Figure Lengend Snippet: HGE-20 cells were incubated for 6 hours with cycloheximide and with or without FCF and geldanamycin. ErbB2 was immunoprecipitated, followed by western blot with antibodies against ubiquitin, and then ErbB2. Ubiquitylation of ErbB2 in the presence of FCF and geldanamycin was greater than with either agent alone (A). Western blot of total cell lysates obtained following the same incubation conditions demonstrates an additive effect of both agents on the level of mature ErbB2. β-actin was used as a loading control (B). HGE-20 cells were incubated with FCF and/or geldanamycin and the indicated inhibitors of protein degradation for 6 hours, followed by western blot using antibodies against ErbB2, with β-actin as a loading control. FCF-induced ubiquitylated forms of ErbB2, shown as an increased density above the main band (arrowheads), are increased by CA074-me in the presence and absence of geldanamycin. CA074-me protected geldanamycin-induced ErbB2 degradation products (indicated by # on the blot) independent of FCF. Lactacystin did not augment the effects of CA074-me on the FCF or geldanamycin-induced changes, demonstrating that proteasomes are not directly involved in ErbB2 degradation (C). HGE-20 cells were incubated with the indicated inhibitors for 6 hours, fixed, and immunofluorescence was completed using antibodies against ErbB2 and LAMP2. Co-localization of ErbB2 with lysosomes was increased in the presence of both FCF and geldanamycin (D). Error bars, s.d., n=3 independent experiments, * - significant difference from no-FCF condition, Student’s t-test, P<0.05, ** - significant difference between indicated conditions, Student’s t-test, P<0.05, C-cycloheximide, GA-geldanamycin, UM-unmodified ErbB2, V-vehicle, CA-CA074-me, L-lactacystin.

Article Snippet: The following primary antibodies were used: ErbB2 (mouse, Ab-17, clone E2-4001+3B5, 1:1000 western blot, 1:100 immunofluorescence, ThermoFisher, Waltham, MA, USA), β-actin (mouse, clone C4, 1:1000, Milipore, Billarica, MA, USA, rabbit, 1:1000, Cell Signaling Technologies, Beverly, MA, USA), septin-2 (Sigma prestige, 1:2000 western blot, 1:100 immunofluorescence), septin-9 (Sigma prestige, 1:1000 western blot, 1:100 immunofluorescnece), septin-7 (rabbit, H-120, Santa Cruz, 1:1000), EGFR (1:1000, NeoMarkers, Fremont, CA), ubiquitin (mouse, clone P4D1, 1:500, Santa Cruz), NSF (mouse, clone NSF-1, 1:1000, Millipore), Na,K-ATPase β subunit (mouse, 1:1000, Affinity Bioreagents, Golden, CO, USA), LAMP2 (rabbit, Sigma prestige, 1:50), PSMC-1 (rabbit, Sigma prestige, 1:50), caveolin-1 (mouse, 1:100 immunofluorescence, BD Transduction Laboratories, Franklin Lakes, NJ, USA), EEA-1 (rabbit, 1:100 immunofluorescence, Abcam, San Francisco, CA, USA), cofilin (rabbit, Abcam, Cambridge, MA, USA, 1:1000), CIN85 (clone 179.1.E1, mouse, Upstate Cell Signaling Solutions, Lake Placid, NY, 1:1000), green fluorescent protein (rabbit, Clontech, Mountain View, CA, USA, 1:1000), human influenza hemagglutinin (HA) (mouse, clone F-7, Santa Cruz, 1:1000).

Techniques: Incubation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control, Immunofluorescence