Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: (A) BioID and BioID2 crystal structures (Protein Data Bank 1BIB and 2EAY) showing the splitting site of split-BioID (E 256 /G 257 ) and its corresponding position in BioID2 (K 171 /S 172 ). (B) Principle of the rapamycin-induced dimerization set-up to test split-BioID fragments: FKBP is fused to the N-terminal fragment and FRB to the C-terminal fragment. PDB is tested in the presence or absence of rapamycin. (C) Blots of lysates of HeLa cells transiently transfected with the indicated constructs and cultivated for 24h in biotin-containing medium in the presence or absence of rapamycin. Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against FLAG and Myc tag as indicated. The asterisk (*) indicates a non-specific cross reactivity.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Transfection, Construct, Labeling, Expressing

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: Blots of lysates of HeLa cells transiently transfected with the indicated constructs and incubate with 50 μM biotin for 24 h (left), 1 h (middle) or 10 min (right). Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against the Myc tag as indicated.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Transfection, Construct, Labeling, Expressing

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: (A) Scheme of the biotinylation assay. microID random variants were surface-presented via Aga1p:Aga2p. The yeast surface was biotinylated in presence of ATP and biotin. The His-tag served as a presentation marker. Biotin was detected with a streptavidin-APC conjugate. (B) Density plots of surface presentation (Y-axis) vs. cell surface biotinylation (X-axis). The error-prone PCR based-yeast surface display library was screened by stepwise decreased biotinylation time. Upper row: Comparison of microID with the initial library (R1) after a 17 h biotinylation assay. Bottom row: same with the Round 3 (R3) library after a 10 min biotinylation assay. (C) Single clone analysis from the Round 3 library. (D) Positions of the mutated residues (structure based on PDB: 3EFR processed with the program ChimeraX). (E) Activity measurement of microID variants with an ELISA-based biotinylation assay. Serum albumin biotinylation mediated by the indicated enzymes was measured through the absorbance at 405 nm and was normalized to the activity of microID (set to 100%). Error bars are standard deviation. N=5; n=3.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Cell Surface Biotinylation Assay, Marker, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: Blots of lysates of HeLa cells transiently transfected with the indicated constructs and incubated with 50 μM biotin for 10 min. Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against the Myc tag as indicated. (A) Comparison microID, ultraID-4 and -5, and TurboID. (B) Comparison microID, microID L41P-only, microID R40G/L41P (= ultraID), microID S24C/R40G/L41P (=ultraID-4 with K169R reverted to WT) and TurboID.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Transfection, Construct, Incubation, Labeling, Expressing

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: (A) Blots of lysates of HeLa cells transiently transfected with the indicated constructs and incubated (+) or not (−) with 50 μM biotin for 24h (left), 1h (middle) or 10 min (right). Biotinylation was analyzed using IRDye680-labeled streptavidin and expression levels of the fusion proteins with antibodies against the Myc tag as indicated. (B) Quantification of (A). Circles and squares of the same color belong to the same replicate experiments.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Transfection, Construct, Incubation, Labeling, Expressing

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: (A) Blots of lysates of S. cerevisiae stains expressing the indicated Asc1 fusion proteins and incubated or not (−) with 10 μM biotin for the indicated times. The fusion proteins were expressed from plasmids in a Δ asc1 strain, and the ASC1 wild-type strain carrying the empty vector served as a control. Biotinylation was analyzed using HRP-labeled streptavidin and expression levels of the fusion proteins with antibodies against Asc1 as indicated. (B) Blots of lysates of E. coli cells transformed with expression plasmids for ultraID, microID or the control unrelated protein CNOT9. The cells had been incubated (+) or not (−) with biotin for 16 h. The asterisk (*) indicate the signal for the endogenous bacterial biotinylated protein BCCP.

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Expressing, Incubation, Plasmid Preparation, Labeling, Transformation Assay

Journal: bioRxiv

Article Title: ultraID: a compact and efficient enzyme for proximity-dependent biotinylation in living cells

doi: 10.1101/2021.06.16.448656

Figure Lengend Snippet: (A) Blots of lysates of P19 cells stably expressing ultraID-Ago2, of P19 cells knock-out for endogenous γ1-COP and rescued by γ1-COP-ultraID, and of P19 cells knock-out for γ2-COP and rescued by γ2-COP-ultraID lines. The fusion proteins were detected with antibodies against γ1- and γ2-COP and biotinylation with IRDye680-labeled streptavidin (B) Experimental setup for the determination of the membrane-associated interactome of γ1-COP and γ2-COP. (C) Volcano plots of the proteins identified by LC-MS/MS with the mock vs. BFA samples. Significant hits (p-value < 0.01 and log 2 fold change > 2 in this analysis, and p-value < 0.01 and log2 fold change > 2 in the γ-COP vs Ago2 comparison under mock conditions), are indicated with gene names in red. (D) Proteins identified as membrane interactors of γ1-COP and γ2-COP (see text).

Article Snippet: The membranes were then incubated with polyclonal rabbit anti-Asc1 (in 5% milk powder, 0.1% Tween 20, PBS) followed by incubation with a peroxidase-coupled secondary antibody or with Pierce™ High Sensitivity Streptavidin-HRP (Thermo Fisher, #21130, diluted 1:2000 in 1% BSA, 0.1% Tween 20, PBS).

Techniques: Stable Transfection, Expressing, Knock-Out, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Overexpression Promotes Mesangial Cell Proliferation and Aggravates Renal Fibrosis of db/m Mice. A) Immunofluorescence and B) its quantitative analysis was used to detect the expression of PCNA, Ki67 in the mesangial cells (PCNA, green fluorescence; Ki67, red fluorescence; Scale bar: 20 µm); n = 3; C) Cell cycle of mesangial cells by flow cytometry. n = 3; D) The protein expression level of Cyclin D, Cyclin E, FN, and COL I in mesangial cells by western blot, n = 3; E) The expressions of LN, FN, COL I and COL IV in mesangial cells by ELISA, n = 5; F) Kidney volume and weight of mice, n = 6; G) BUN, albumin, UACR and α‐MG of mice, n = 6; H) PAS staining (Positive area: red; Scale bar: 20 µm) and transmission electron microscopy (Scale bar: 2 µm) of kidney tissue in mice; I) Sirius red staining (Positive area: red) and Masson staining (Positive area: blue) of kidney tissue in mice, (Scale bar: 20 µm); J) The expression of FN and Collagen I in glomeruli by immunohistochemical staining (Positive area: brown; Scale bar: 20 µm); K,L) The quantitation of Masson and Sirius red postive staining areas, n = 6; M,N) The quantification of Fibronectin and Collagen I expression, n = 6. NG: normal mesangial cell; OV: normal mesangial cell with vector; OE: normal mesangial cell with GABPα/β overexpression lentivirus; db/m : normal control mice; db/m +Veh: db/m mice with vector; db/m +OE: db/m mice with intra‐renal injection of GABPα/β overexpression adeno‐associated virus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to OV or db/m + V eh.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Over Expression, Immunofluorescence, Expressing, Fluorescence, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Transmission Assay, Electron Microscopy, Immunohistochemical staining, Quantitation Assay, Plasmid Preparation, Control, Injection, Virus

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Knockdown Inhibits Mesangial Cell Proliferation under High Glucose Conditions and Ameliorates Renal Fibrosis of db/db Mice. A) Immunofluorescence and B) its quantitative analysis was used to detect the expression of PCNA, Ki67 in the mesangial cells (PCNA, green fluorescence; Ki67, red fluorescence; Scale bar: 20 µm), n = 3. C) Cell cycle of mesangial cells by flow cytometry. n = 3. D) The protein expression levels of Cyclin D, Cyclin E, FN, and COL I in mesangial cells by western blot, n = 3. E) The expressions of LN, FN, COL I, and COL IV by ELISA, n = 5. F) Kidney volume and weight of mice, n = 6. G) BUN, albumin, UACR, and α‐MG of mice, n = 6. H) PAS staining (Positive area: red Scale bar: 20 µm) and transmission electron microscopy (Scale bar: 2 µm) of kidney tissue in mice. I) Sirius red staining (Positive area: red) and Masson staining (Positive area: blue) of kidney tissue in mice, (Scale bar: 20 µm). J) The expression of FN and Collagen I in glomeruli by immunohistochemical staining (Positive area: brown; Scale bar: 20 µm). K,L) The quantitation of Masson and Sirius red postive staining areas, n = 6. M,N) The quantification of Fibronectin and Collagen I expression, n = 6. NG: normal mesangial cell; HG: mesangial cell with 30 mM glucose; HKV: high glucose cultured mesangial cell with vector; HKD: high glucose cultured mesangial cell with GABPβ‐knockdown lentivirus; db/m : normal control mice; db/db : diabetic model mice; db/db +veh: db/db mice with intra‐renal injection of vector; db/db +KD: db/db mice with intra‐renal injection of GABPβ‐knockdown adeno‐associated virus. Data are expressed as mean ± s.e.m. Statistical significance was assessed using a one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to NG or db/m , # p < 0.05, ## p < 0.01, compared to HKV or db/db + veh.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Knockdown, Immunofluorescence, Expressing, Fluorescence, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Transmission Assay, Electron Microscopy, Immunohistochemical staining, Quantitation Assay, Cell Culture, Plasmid Preparation, Control, Injection, Virus

Journal: Advanced Science

Article Title: GABP Promotes Mesangial Cell Proliferation and Renal Fibrosis Through GLI1 in Diabetic Nephropathy

doi: 10.1002/advs.202407462

Figure Lengend Snippet: GABP Regulates Cell Proliferation and ECM by Inducing GLI1 Expression in Mesangial Cells. A–C) The mRNA expression levels of Collagen I, Fibronectin, and Collagen IV, n = 3; D) The protein expression levels of Collagen I and Fibronectin, n = 3; E,F) The accumulation of Fibronectin and Collagen I in cells was detected by immunofluorescence (DAPI, blue fluorescence; Fibronectin/Collagen I, red fluorescence; Scale bar: 20 µm). NG: normal mesangial cell group (5.56 mmol L −1 glucose concentration); HG: mesangial cell with 30 mM glucose group; HG+DMSO: high glucose cultured mesangial cell with DMSO control group; HG+GANT61: high glucose cultured mesangial cells with 10 µM GANT61 group; GABP: normal mesangial cells with GABPα/β‐overexpression lentivirus; GABP+DMSO: normal mesangial cells with GABPα/β‐overexpression lentivirus and DMSO group; GABP+GANT61: normal mesangial cells with GABPα/β‐overexpression lentivirus and 10 µM GANT61 group; Data are expressed as mean ± s.e.m. Statistical significance was assessed using one‐way ANOVA with Tukey's test, * p < 0.05, ** p < 0.01, compared to the NG group, # p <0.05, ## p <0.01, compared to the HG group, @ p < 0.05, @@ p < 0.01, compared to the GABP group.

Article Snippet: The following antibodies were used: GABPα (21542‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:50, IF/ICC: 1:200), GABPβ (12597‐1‐AP, Proteintech, WB: 1:1000, IHC: 1:50, IF/ICC: 1:500), GLI1 (66905‐1‐Ig, Proteintech, WB: 1:5000, IF/ICC: 1:400) and Fibronectin (15613‐1‐AP, Proteintech, WB: 1:2000, IHC: 1:2000, IF/ICC: 1:50) for Western blotting, IHC and IF staining; β‐actin (AP0060, Bioworld, WB:1:5000, IF: 1:500), Cyclin D1 (26939‐1‐AP, Proteintech, WB: 1:5000, IHC: 1:750, IF/ICC: 1:400), Cyclin E1 (11554‐1‐AP, Proteintech, WB: 1:500, IHC: 1:400) for Western blotting; Cyclin D1(A11022, ABclonal, WB: 1:500, IHC: 1:50, IF/ICC: 1:50), Cyclin E (120039, absin, WB: 1:500, IHC‐P: 1:100, IHC‐F: 1:100, IF: 1:100), PCNA (13110, Cell Signaling Technology, WB: 1:1000, IHC: 1:4000, IF: 1:400), KI67 (AF0198, Affinity, WB: 1:500, IHC: 1:50, IF/ICC: 1:100) and PDGF Receptor β (#3169, CST, WB: 1:1000, IHC: 1:50, IF: 1:100) for IF staining; COL1A1 (67288‐1‐Ig, Proteintech, WB: 1:5000, IHC: 1:2500, IF:1:200) for IF staining and Western blotting.

Techniques: Expressing, Immunofluorescence, Fluorescence, Concentration Assay, Cell Culture, Control, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Metformin Promotes AMP-activated Protein Kinase-independent Suppression of ΔNp63α Protein Expression and Inhibits Cancer Cell Viability *

doi: 10.1074/jbc.M116.769141

Figure Lengend Snippet: ΔNp63α and fibronectin are critical in metformin-induced cell detachment and cell death. A, FaDu cells were cultured in 24-well plates coated with 1% agarose for the indicated time. An MTS assay was performed for cell viability. OD, optical density. B, FaDu cells stably expressing shRNA against GFP or p63 were subjected to Q-PCR analysis for integrin β4 (ITGB4), fibronectin 1 (FN1), E-cadherin (E-cad), and N-cadherin (N-cad). C–E, expression of ΔNp63α failed to rescue metformin (Met)-induced cell detachment and death. C, whole-cell lysates from FaDu cells stably expressing HA/FLAG-ΔNp63α were subjected to Western blotting. FaDu cells stably expressing HA/FLAG-ΔNp63α or vector (Vec) was treated with or without metformin (10 mm) for 24 h under 1.0 mg/ml glucose (Glu). Attached cells were determined (D), and cell viability was measured using an MTS assay (E). F, FaDu cells stably expressing HA/FLAG-ΔNp63α was treated with or without metformin (10 mm) for 24 h under 1.0 mg/ml glucose and subjected to Western blotting analyses as indicated. G and H, FaDu cells were seeded on tissue culture plates coated with either 50 μg/ml fibronectin, collagen 1, or BSA. Cells were then cultured for 36 h in the presence or absence of metformin (5 or 10 mm) under 1.0 mg/ml glucose. Quantitation of attached cells is presented (G); cell viability was determined using an MTS assay (H). I and J, FaDu cells stably expressing ΔNp63α or vector control were seeded on tissue culture plates coated with or without fibronectin. Cells were then cultured in the presence or absence of 10 mm metformin in DMEM containing 1.0 mg/ml glucose for 36 h. Microscopic images of the cells and attached cells are presented (I), and cell viability was determined using an MTS assay (J). Data are presented as means ± S.E. from three independent experiments in triplicates. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant.

Article Snippet: Antibodies for WWP1 (ab104440, 1:10,000), Integrin β1 (ab52971, 1:1000), ITCH (ab109018, 1:1000), or fibronectin (ab6328, 1:2000) were purchased from Abcam (Cambridge, MA).

Techniques: Cell Culture, MTS Assay, Stable Transfection, Expressing, shRNA, Western Blot, Plasmid Preparation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Metformin Promotes AMP-activated Protein Kinase-independent Suppression of ΔNp63α Protein Expression and Inhibits Cancer Cell Viability *

doi: 10.1074/jbc.M116.769141

Figure Lengend Snippet: Schematic of metformin-mediated anti-cancer activity. Glucose deprivation or 2-DG promotes metformin-mediated down-regulation of ΔNp63α and its downstream target fibronectin, leading to disruption of cell matrix adhesion and subsequent apoptosis.

Article Snippet: Antibodies for WWP1 (ab104440, 1:10,000), Integrin β1 (ab52971, 1:1000), ITCH (ab109018, 1:1000), or fibronectin (ab6328, 1:2000) were purchased from Abcam (Cambridge, MA).

Techniques: Activity Assay