Journal: Protein Science : A Publication of the Protein Society

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1002/pro.4859

Figure Lengend Snippet: Expression of the YPEL family genes in cell lines. (a) Based on data from the DepMap portal, the average expression of the YPEL gene family members relative to the expression of YPEL1 in breast cancer cell lines (DM‐BCCL) or only in MCF7 cells (DM‐MCF7) is shown. For comparison, our RT‐qPCR analyses for the relative expression of YPEL genes in MCF7 cells (MCF7) are also indicated. (b) To assess whether or not E2‐ERα signaling is involved in the regulation of YPEL2 , 3 , and 5 expressions, MCF7 cells grown for 72 h in CD‐FBS containing growth medium were treated without (EtOH as control) or with 10 −9 M E2, and/or 10 −9 M ICI, a complete ER antagonist, for 24 h. cDNAs generated from total RNA were subjected to qPCR. We also used the E2‐ERα responsive gene TFF1 as a control. Star indicates a significant change compared to EtOH control. (c) Relative to YPEL1 , the expression of YPEL genes in COS7 cells was assessed by qPCR using cDNA from total RNA obtained from COS7 cells grown in steady‐state conditions. (d) To evaluate the synthesis of YPEL1‐5 proteins, which Y3.1 and Y3.2 indicate short and long variant YPEL3 proteins, COS7 cells were transiently transfected with the expression vector pcDNA3.1(−) bearing none (EV) or cDNA for a YPEL protein. Total protein extracts of COS7 cells grown in steady‐state condition were subjected to SDS‐15%PAGE followed by WB using a pan‐YPEL antibody (SCBT, sc99727) and an HRP‐conjugated goat‐anti‐rabbit secondary antibody (Advansta R‐05072–500). Membranes were re‐probed with an antibody specific to HDAC1 (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. (e) To assess the synthesis of endogenous YPEL proteins in COS7 cells, protein extracts ranging from 50 to 150 μg were subjected to WB using the pan‐YPEL antibody. We also used protein extracts from COS7 cells transiently transfected with pcDNA3.1(−) bearing the 3F‐YPEL2 cDNA for comparison. Membranes were re‐probed with the HDAC1 antibody (Abcam, ab19845). Molecular masses (MM) in kDa are indicated. (f) To assess the intracellular localization of endogenous YPEL (YPEL) proteins, COS7 cells grown on coverslips were subjected to ICC using the pan‐YPEL antibody followed by an Alexa Fluor 594‐conjugated goat anti‐rabbit secondary antibody (Abcam, ab150080) or a vimentin (sc‐6260) antibody followed by an Alexa Fluor 488‐conjugated goat anti‐mouse secondary antibody (Abcam, ab150113). DAPI was used to indicate the nucleus. The scale bar is 20 μm. (g,h) The intracellular localization of 3F‐YPEL2 was evaluated in transiently transfected cells with the use of the Flag (g) or the pan‐YPEL (h) antibody or GFP fusion (i). DAPI staining indicates the nucleus. The scale bar is 20 μm.

Article Snippet: To obtain time‐dependent profiling of stable, transient, and/or weak protein interactions of YPEL2, we initially generated a TurboID‐HA cDNA using the 3xHA‐TurboID‐NLS‐pcDNA3 (Branon et al., ) expression vector (Plasmid #107171; Addgene, Massachusetts, USA) as the template.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Generated, Variant Assay, Transfection, Plasmid Preparation, Staining

Journal: Protein Science : A Publication of the Protein Society

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1002/pro.4859

Figure Lengend Snippet: Interaction of 3F‐YPEL2 and HA‐ELAVL1. (a) To assess the intracellular localization of 3F‐YPEL2 and/or HA‐ELAVL1, COS7 cells grown on coverslips un‐transfected (UT) or transiently transfected for 36 h with the expression vector pcDNA3.1(−) bearing the 3F‐YPEL2 or HA‐ELAVL1 cDNA were stained with the Flag or the HA antibody. DAPI was used to indicate the nucleus. The scale bar is 20 μm. (b) COS7 cells were transfected (+) with the expression vector bearing 3F‐YPEL2 and/or HA‐ELAVL1 cDNA to examine the protein synthesis. The synthesis of proteins was assessed by WB using the Flag or the HA antibody. HDAC1, used as a loading control, was probed with the HDAC1 antibody. (c) The cellular extracts (500 μg) of transiently co‐transfected COS7 cells were subjected to Co‐IP with the HA, Flag, or isotype‐matched IgG. Fifty micrograms of lysates was used as input control. The precipitates were subjected to SDS‐10%PAGE followed by WB using the Flag or the HA antibody. Molecular masses (MM) in kDa are indicated. (d) To assess in cellula interaction of 3F‐YPEL2 and HA‐ELAVL1, the proximity ligation assay was carried out in transiently transfected COS7 cells grown on coverslips. Cells were fixed, permeabilized, blocked, and probed with the HA and/or the Flag antibody. Cells were then subjected to fluorescent probes for circular DNA amplification for proximity interaction foci. DAPI was used for nuclear staining. Images were captured with a fluorescence microscope. The scale bar is 20 μm. (e) The intracellular localization, (f) protein synthesis, (g) interaction, and (h) PLA of 3F‐YPEL2 and/or HA‐SQSTM1 in transiently transfected COS7 cells were assessed as described in the legend of a–d. HDAC1 was used as a loading control.

Article Snippet: To obtain time‐dependent profiling of stable, transient, and/or weak protein interactions of YPEL2, we initially generated a TurboID‐HA cDNA using the 3xHA‐TurboID‐NLS‐pcDNA3 (Branon et al., ) expression vector (Plasmid #107171; Addgene, Massachusetts, USA) as the template.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Amplification, Fluorescence, Microscopy

Journal: Protein Science : A Publication of the Protein Society

Article Title: Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

doi: 10.1002/pro.4859

Figure Lengend Snippet: Stress granule formation in COS7 cells in response to sodium arsenite (SA). (a) To assess the formation of stress granules, COS7 cells grown on coverslips were treated without (−SA) or with 200 μM sodium arsenite (+SA) for 1 h. Cells were fixed and subjected to hybridization‐couple ICC using an ATTO488 conjugated oligo‐dT probe and the polyclonal G3BP1 antibody (ab200550) followed by an Alexa Fluor‐647 conjugated secondary antibody. DAPI indicates the nucleus. The scale bar is 20 μm. (b) To assess the co‐localization of the endogenous YPEL proteins (YPEL) with G3BP1, cells treated with SA for 1 h were fixed and subjected to ICC using the monoclonal G3BP1 antibody (SCBT, sc‐365338) followed by an Alexa Fluor‐488 conjugated secondary antibody (405319, Biolegend) and the pan‐YPEL antibody (SCBT, sc99727) followed by an Alexa Fluor‐647 conjugated goat‐rabbit secondary antibody (Abcam, ab150083). DAPI was used to indicate the nucleus. (c) To assess whether 3F‐YPEL2 also co‐localizes with G3BP1 in SGs, cells transfected with the expression vector pcDNA3.1(−) bearing 3F‐YPEL2 cDNA were subjected to ICC using the polyclonal G3BP1 antibody (Abcam, ab200550) followed by an Alexa Fluor‐647 conjugated secondary antibody and the monoclonal Flag antibody (Sigma‐Aldrich, F1804) followed by an Alexa Fluor 488‐conjugated secondary antibody. DAPI indicates the nucleus. (d) To examine the co‐localization of YPEL and G3BP1 during the formation and disassembly of SGs, COS7 cells grown on coverslips were treated with 200 μM sodium arsenite (+SA) for 0, 15 min, 30 min 1 h, and 2 h. (e) Following 1 h of 200 μM sodium arsenite (+SA) treatment, cells (post‐stress, PS) were washed and incubated in the growth medium without SA for 0, 1, 2, 3, or 4 h. (d and e) Fixed cells were then subjected to ICC using the monoclonal G3BP1 antibody (sc‐365338) and the pan‐YPEL antibody (sc99727) followed by secondary antibodies for visualization with a fluorescence microscope. DAPI staining indicates the nucleus. The scale bar is 20 μm.

Article Snippet: To obtain time‐dependent profiling of stable, transient, and/or weak protein interactions of YPEL2, we initially generated a TurboID‐HA cDNA using the 3xHA‐TurboID‐NLS‐pcDNA3 (Branon et al., ) expression vector (Plasmid #107171; Addgene, Massachusetts, USA) as the template.

Techniques: Hybridization, Transfection, Expressing, Plasmid Preparation, Incubation, Fluorescence, Microscopy, Staining

Journal: Cancer Cell International

Article Title: DFNA5 regulates immune cells infiltration and exhaustion

doi: 10.1186/s12935-022-02487-0

Figure Lengend Snippet: Correlation analysis between DFNA5 and relate genes and markers of immune cells in TIMER

Article Snippet: A pcDNA3.0-flag, pcDNA3.0-PDCD1-3x-myc and pcDNA3.0-CD274-3x-myc plasmid expression vector were obtained from our lab. A pcDNA3-based expression vector for TurboID was obtained from addgene(Addgene: #107169).

Techniques:

Journal: Cancer Cell International

Article Title: DFNA5 regulates immune cells infiltration and exhaustion

doi: 10.1186/s12935-022-02487-0

Figure Lengend Snippet: Correlation of DFNA5 expression with tumour-infiltrating lymphocytes in COAD (colon adenocarcinoma), LIHC (hepatocellular carcinoma), and LUAD (lung adenocarcinoma). A DFNA5 expression is significantly negatively related to tumour purity and has significant positive correlations with infiltrating levels of CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells in COAD but not B cells. B DFNA5 expression has significant correlations with tumour purity and infiltrating levels of B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells in LIHC. C DFNA5 expression is significantly negatively related to tumour purity and has significant positive correlations with infiltrating levels of B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells in LUAD. D The increased content of Flag-DFNA5 plasmids co-transfected with 3x-myc-PDCD1 or 3x-myc-CD274 plasmid, the expression levels of FLAG, 3x-myc and β-actin were detected by western blot. The band grey value of the rightmost well of 3x-myc-PDCD1 or 3x-myc-CD274 relative to the corresponding beta-actin were set at 1.0. The other treatments were compared with them

Article Snippet: A pcDNA3.0-flag, pcDNA3.0-PDCD1-3x-myc and pcDNA3.0-CD274-3x-myc plasmid expression vector were obtained from our lab. A pcDNA3-based expression vector for TurboID was obtained from addgene(Addgene: #107169).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: mBio

Article Title: Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling

doi: 10.1128/mbio.03336-24

Figure Lengend Snippet: Cell surface labeling with TurboID fusion proteins to a protein toxin. ( A ) Recombinant TurboID fusions to fragments of TcdB, purified from E. coli . ( B ) Cell surface labeling with TurboID. Recombinant protein was incubated on confluent cells with supplemental ATP, MgCl 2 , and biotin for an hour at 37°C. ( C ) Average spectral count for prey proteins with BFDR ≤ 0.01 of bait TurboID-TcdB ΔGTD on HEK293T cells.

Article Snippet: E. coli expression optimized TurboID (Addgene Catalogue #107177) from the lab of Alice Ting was cloned into a Champion pET-SUMO vector (Invitrogen) alone or as fusions with DT or TcdB using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Labeling, Recombinant, Purification, Incubation

Journal: mBio

Article Title: Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling

doi: 10.1128/mbio.03336-24

Figure Lengend Snippet: SAINT scoring for prey protein LRP1 labeled on 18Co cells by the recombinant bait proteins TurboID-TcdB ΔGTD , TcdB CROP , and TurboID-TcdB 544–1,832

Article Snippet: E. coli expression optimized TurboID (Addgene Catalogue #107177) from the lab of Alice Ting was cloned into a Champion pET-SUMO vector (Invitrogen) alone or as fusions with DT or TcdB using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Labeling, Recombinant

Journal: mBio

Article Title: Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling

doi: 10.1128/mbio.03336-24

Figure Lengend Snippet: dtR-TurboID fusion labels DT receptor HBEGF. ( A ) Recombinant N- and C-terminal fusions of dtR and TurboID biotinylate soluble HBEGF. In vitro assay of 300 nM enzyme with and without 50 µM biotin and 20 µM HBEGF was analyzed by western blotting to detect biotinylation signal. TurboID-toxin chimeras were expressed with a C-terminal myc tag. ( B ) dtR-TurboID labels HBEGF on ARN8:HBEGF + cells. One hundred fifty nanomolar of recombinant TurboID or dtR-TurboID was incubated with suspended HBEGF KO (ARN8:HBEGF - ) or HBEGF-expressing (ARN8:HBEGF + ) ARN8 cells with 50 µM biotin for 10 minutes at room temperature (RT). Self-biotinylation is indicated with an asterisk. ( C ) High-confidence prey proteins identified with BFDR ≤ 0.01 threshold performed on three ARN8 cell lines by dtR-TurboID: HBEGF-overexpressing (OE), wild-type (WT), and HBEGF KO (KO). Data shown are from two replicates on OE and KO cells and a single replicate on WT cells.

Article Snippet: E. coli expression optimized TurboID (Addgene Catalogue #107177) from the lab of Alice Ting was cloned into a Champion pET-SUMO vector (Invitrogen) alone or as fusions with DT or TcdB using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Recombinant, In Vitro, Western Blot, Incubation, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila

doi: 10.1007/s00018-023-04778-9

Figure Lengend Snippet: Ddrgk1 mediates Atg9 Ufmylation and stabilizes its proteins. a Immunoblots to monitor the transient interaction between Ddrgk1-TurboID-V5 and Flag-Atg9. S2 cells were transfected to express TurboID-V5 or Ddrgk1-TurboID-V5 with Flag-Atg9. Cells were treated with biotin or water as a control (-) as indicated. Biotin-labeled proteins were recovered using Streptavidin beads, and blots were probed with anit-V5 and anti-Flag to visualize Ddrgk1 and bound Atg9. Relative protein levels were quantified and calculated for representative lanes. b Immunoblots to monitor Ufmylation of Atg9-GFP expressed under elav-Gal4 control in aged (21-day-old) fly heads. Left panel: Atg9-GFP proteins were recovered using GFP antibody raised in Alpaca, and blots were probed with anti-Ufm1 to visualize Ufmylation. Alpaca IgG was used as a control. Right panel: Atg9-GFP proteins were recovered from aged heads expressing UAS-Ddrgk1RNAi under elav-Gal4 control. Anti-β-actin was used as loading control. c Immunoblots to visualize the level of GFP tagged Atg9 expressed in 21-day-old fly brains with and without Ddrgk1 depletion. Lower panel: histogram showing the normalized ratio of GFP/β-actin indicated for representative lanes in upper panel. Data represent 3 independent experiments. **p < 0.01. d Correlation analysis of Ddrgk1 versus Atg9A in 7255 human samples from 6805 patients with tumors in CNS/brain using cBioPortal (www.cbioportal.org). Pearson correlation coefficient rho = 0.71 (p < 0.001). e Upper panel: immunoblots to visualize the levels of endogenous Atg9A and Ddrgk1 in MEF cells harvested from the Ddrgk1F/F:ROSA26-CreERT2 mice. MEFs were treated with 4-OHT or ethanol to induce Ddrgk1 deletion or as a control. Before harvested, MEFs were pretreated with cycloheximide (CHX) for the indicated times. Anti-β-actin was used as loading control. Lower panel: histogram showing the quantification of Atg9A levels normalized to β-actin. f Immunoblots to monitor Ufmylation of Atg9A-V5. MEFs were treated with 4-OHT to induce Ddrgk1 deletion. Atg9A-V5 proteins were recovered using V5 antibody from MEFs transfected to express Atg9A-V5. Blot was probed with anti-Ufm1 to visualize Atg9A Ufmylation. Anti-β-actin was used as loading control

Article Snippet: TurboID A V5 tag was added to a TurboID ligase derived from Flag-TurboID (addgene 124646) by PCR before the entire DNA fragment was cloned into pUAST-attB vector at EcoRI and XhoI sites for fly expression.

Techniques: Western Blot, Transfection, Control, Labeling, Expressing

Journal: bioRxiv

Article Title: Proximity labelling and evolutionary evidence reveal insights into IL-1α nuclear networks and function

doi: 10.1101/2023.06.26.546506

Figure Lengend Snippet: ( A ) Schematic of pro-IL-1α, pro-IL-1α-TurboID (pro-IL-1α-TID) and TurboID (TID) constructs. ( B ) HeLa cells were transfected with constructs in (A), then treated with biotin (500 µM, 30 min), and analysed by immunofluorescence microscopy (n=2). Anti-IL-1α labels pro-IL-1α and mature IL-1α, anti-BirA labels TurboID, streptavidin-HRP labels biotinylated proteins. Dark blue represents nuclei stained by DAPI. Scale bars are 10 µm. ( C, D ) HeLa cells were untransfected (UT) or transfected with constructs in (A), then treated with biotin (500 µM, 30 min). Cell lysates were probed for (C) IL-1α (anti-IL-1α), TurboID (anti-BirA) (n=4), and (D) biotin (StrepHRP) (n=4). ( E ) HeLa cells were untransfected (UT) or transfected with pro-IL-1α or pro-IL-1α-TID and were then treated with ionomycin (Ion) for 0, 5, 10, 15, 30 or 60 minutes (n=4). Cell lysates were probed for IL-1α by western blotting. 0 and 60 minutes were used as representatives for UT cells.

Article Snippet: To generate the TID expression vector, the TurboID sequence was amplified from Addgene plasmid #107173 using primers tF1F and tF1R.

Techniques: Construct, Transfection, Immunofluorescence, Microscopy, Staining, Western Blot

Journal: bioRxiv

Article Title: Proximity labelling and evolutionary evidence reveal insights into IL-1α nuclear networks and function

doi: 10.1101/2023.06.26.546506

Figure Lengend Snippet: ( A ) Schematic illustrating the pipeline of biotinylation enrichment analysis by mass spectrometry. HeLa cells were transfected with pro-IL-1α-TurboID (Alpha-TID) or TurboID (TID), and then treated with biotin (500 µM, 30 min, n=4). Streptavidin pull-down of biotinylated proteins was performed, and biotinylated proteins were analysed by mass spectrometry. ( B ) Principal component analysis of LFQ intensities. A single square denotes an independent experiment. Experimental groups (Alpha-TID or TID) are grouped by colour. ( C ) Volcano plot of proteins enriched in Alpha-TID (blue) compared to TID following two-sample t-test of LFQ intensity values (significance determined as s0=2; FDR=0.01). ( D-G ) Ingenuity pathway analysis (IPA) of proteins significantly enriched in Alpha-TID. ( D ) Subcellular location of all significantly enriched proteins. ( E ) Nuclear location of significantly enriched nuclear proteins. ( F ) Canonical pathways (p<0.01) and ( G ) biological functions (top 10) associated with proteins significantly enriched in Alpha-TID. Black bars represent log 10 (p-value) of overlap between Alpha-TID proteins and the proteins within each canonical pathway or biological function and were obtained via the right-tailed Fisher’s exact test implemented in the IPA software. Orange line denotes number of Alpha-TID proteins present within each canonical pathway or biological function.

Article Snippet: To generate the TID expression vector, the TurboID sequence was amplified from Addgene plasmid #107173 using primers tF1F and tF1R.

Techniques: Mass Spectrometry, Transfection, Software

Journal: bioRxiv

Article Title: Proximity labelling and evolutionary evidence reveal insights into IL-1α nuclear networks and function

doi: 10.1101/2023.06.26.546506

Figure Lengend Snippet: STRING network analysis (generated via https://string-db.org/ ) of proteins significantly enriched in pro-IL-1α-TurboID. Lines represent previously recorded physical interactions identified by STRING, unconnected proteins have no previously recorded interactions with significantly enriched proteins. Histone acetyltransferase (HAT) proteins were identified through gene ontology (GO) analysis. Proteins are displayed using gene symbol label. Gene symbol for the target protein (IL1A; pro-IL-1α) represents the target protein of this experiment.

Article Snippet: To generate the TID expression vector, the TurboID sequence was amplified from Addgene plasmid #107173 using primers tF1F and tF1R.

Techniques: Generated