Journal: Applied and Environmental Microbiology

Article Title: Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

doi: 10.1128/aem.02919-08

Figure Lengend Snippet: FIG. 2. Mutation of the welE gene in L. rhamnosus GG reduces the level of CW-PS. (A) TEM analysis of L. rhamnosus GG wild-type and welE mutant CMPG5351 strains grown in AOAC medium. For wild-type L. rhamnosus GG, the cell-bound EPS layer is indicated. While this layer is absent in the welE mutant CMPG5351, some patches of presumably polysaccharide accumulations could still be detected (black arrow). Additionally, fimbria-like appendages appear to become exposed in welE mutant cells (white arrow). (B) CW-PS were extracted from cell pellets from wild-type L. rhamnosus GG (WT), welE mutant CMPG5351, and the complemented strain CMPG5354. Error bars indicate standard deviations.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristics Reference or source E. coli strains DH5 F 80dlacZ M15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK mK ) supE44 thi-1 girA96 relA1 Gibco-BRL LE392 hsdR574 (rK mK ) supE44 supF58 lacY1 or (lac1ZY)6 galK2 galT22 metB1 trpR55 mcrB ATCC 33572 L. rhamnosus GG strains Wild type Human isolate; wild-type strain GG ATCC 53103 CMPG5351 welE knockout mutant of strain GG; welE::tet(M) This work CMPG5354 CMPG5351 complemented by genomic integration of pCMPG5353 This work Plasmids pFAJ5301 Cloning vector, pUC18 derivative, Eryr 30 pMD5057 Plasmid from L. plantarum 5057 containing a tet(M) marker 11 pEM40 pUC19E-derived integration vector (attB located at the 3 end of tRNALeu locus) containing a 1.6-kb int-attP cassette of phage A2, Apr Eryr 2 pCMPG5351 pFAJ5301 derivative used to inactivate welE gene by insertion of a tet(M) marker via double homologous recombination (for details, see text) This work pCMPG5353 pEM40 derivative containing a fragment with the welE gene in the Ecl136II/EcoRI site (amplified with primers Pro-0722 and Pro-0723), Apr Eryr This work on M arch 11, 2015 by U niversity of P ittsburgh H S LS http://aem .asm .org/ D ow nloaded from in the supplemental material).

Techniques: Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

doi: 10.1128/aem.02919-08

Figure Lengend Snippet: FIG. 3. Mutation of the welE gene in L. rhamnosus GG specifically attenuates the high-molecular-mass, galactose-rich EPS molecules. (A) Analysis of the sugar monomer compositions of the CW-PS extracts of wild-type L. rhamnosus GG (WT), welE mutant CMPG5351, and the complemented strain CMPG5354. The data are expressed in relative amounts, taking the total amount of detected monomeric sugars as 100%. (B) Polyacrylamide gel electrophoresis of the CW-PS extract of wild-type L. rhamnosus GG, welE mutant CMPG5351, and complemented strain CMPG5354. Equal amounts of material were loaded. The upper band corresponds to polysaccharides with a polymer size of 1.4 106 Da based on size exclusion chromatography (data not shown). (C) Detection of individual galactose-rich polysaccharides on wild-type L. rhamnosus GG and welE mutant CMPG5351 by SMFS with PA-1 lectin-functionalized tips. Histograms (n 1,024) of adhesion forces (left) and rupture distances (right) are shown.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristics Reference or source E. coli strains DH5 F 80dlacZ M15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK mK ) supE44 thi-1 girA96 relA1 Gibco-BRL LE392 hsdR574 (rK mK ) supE44 supF58 lacY1 or (lac1ZY)6 galK2 galT22 metB1 trpR55 mcrB ATCC 33572 L. rhamnosus GG strains Wild type Human isolate; wild-type strain GG ATCC 53103 CMPG5351 welE knockout mutant of strain GG; welE::tet(M) This work CMPG5354 CMPG5351 complemented by genomic integration of pCMPG5353 This work Plasmids pFAJ5301 Cloning vector, pUC18 derivative, Eryr 30 pMD5057 Plasmid from L. plantarum 5057 containing a tet(M) marker 11 pEM40 pUC19E-derived integration vector (attB located at the 3 end of tRNALeu locus) containing a 1.6-kb int-attP cassette of phage A2, Apr Eryr 2 pCMPG5351 pFAJ5301 derivative used to inactivate welE gene by insertion of a tet(M) marker via double homologous recombination (for details, see text) This work pCMPG5353 pEM40 derivative containing a fragment with the welE gene in the Ecl136II/EcoRI site (amplified with primers Pro-0722 and Pro-0723), Apr Eryr This work on M arch 11, 2015 by U niversity of P ittsburgh H S LS http://aem .asm .org/ D ow nloaded from in the supplemental material).

Techniques: Mutagenesis, Polyacrylamide Gel Electrophoresis, Polymer, Size-exclusion Chromatography

Journal: Applied and Environmental Microbiology

Article Title: Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase

doi: 10.1128/aem.02919-08

Figure Lengend Snippet: FIG. 4. Biofilm formation and adhesion to mucus and Caco-2 cells by wild-type L. rhamnosus GG (WT), the EPS welE mutant CMPG5351, and complemented strain CMPG5354. (A) Biofilm for- mation (in AOAC medium) is expressed relative to the amount formed by wild-type L. rhamnosus GG. (B) The adhesion capacity to mucus is expressed relative to wild-type L. rhamnosus GG (set as 100%), of which ca. 22% of the added cells adhered. (C) The adhesion capacity to Caco-2 cells is expressed relative to wild-type L. rhamnosus GG, of which ca. 4% of the added cells adhered. Error bars indicate standard deviations.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristics Reference or source E. coli strains DH5 F 80dlacZ M15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK mK ) supE44 thi-1 girA96 relA1 Gibco-BRL LE392 hsdR574 (rK mK ) supE44 supF58 lacY1 or (lac1ZY)6 galK2 galT22 metB1 trpR55 mcrB ATCC 33572 L. rhamnosus GG strains Wild type Human isolate; wild-type strain GG ATCC 53103 CMPG5351 welE knockout mutant of strain GG; welE::tet(M) This work CMPG5354 CMPG5351 complemented by genomic integration of pCMPG5353 This work Plasmids pFAJ5301 Cloning vector, pUC18 derivative, Eryr 30 pMD5057 Plasmid from L. plantarum 5057 containing a tet(M) marker 11 pEM40 pUC19E-derived integration vector (attB located at the 3 end of tRNALeu locus) containing a 1.6-kb int-attP cassette of phage A2, Apr Eryr 2 pCMPG5351 pFAJ5301 derivative used to inactivate welE gene by insertion of a tet(M) marker via double homologous recombination (for details, see text) This work pCMPG5353 pEM40 derivative containing a fragment with the welE gene in the Ecl136II/EcoRI site (amplified with primers Pro-0722 and Pro-0723), Apr Eryr This work on M arch 11, 2015 by U niversity of P ittsburgh H S LS http://aem .asm .org/ D ow nloaded from in the supplemental material).

Techniques: Mutagenesis

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 1 MKRN1 is highly expressed and associated with poor prognosis in patients with CRC. A The expression distribution of mRNA in different tumour cell lines. B The distribution of MKRN1 expression in tumour and normal tissues. C MKRN1 expression in CRC tumour and adjacent non-tumour tissues was verified by WB analysis. D Immunohistochemistry detection of MKRN1 expression in tissue sections of patients with CRC and colitis showing typical photographs (scale bars are 100 and 50 µm, respectively). E The Kaplan–Meier Plotter in the R2 Genomics Analysis Platform was used to draw the overall survival curve. F WB analysis of MKRN1 expression levels in CRC cells (HT29, HCT116, HCT15, and RKO) and normal human colonic fibroblasts (CCD-18Co). ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Expressing, Immunohistochemistry

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 2 MKRN1 has a role in CRC cell proliferation, migration, and invasion. A–C WB analysis of MKRN1 transfection rates in HCT116, HT29, and HCT15 cells. D, E CCK-8 assay for CRC cell viability. F, G Colony formation assay to detect CRC cell proliferation. H, I Migration ability of CRC cells with different MKRN1 expression levels detected using a wound healing assay (Scale bar: 100 µm). G-K Transwell assays for migration and invasion of MKRN1 knockdown and overexpressing cells (Scale bar: 50 µm). L, M Microscopic observation of the morphology of the cell lines HCT116 (Control, sh1-MKRN1) and HCT15 (Vector, OE-MKRN1) (Scale bar: 100 µm). N, O Comparison of epithelial and mesenchymal marker expression following knockdown and MKRN1 overexpression. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Migration, Transfection, CCK-8 Assay, Colony Assay, Expressing, Wound Healing Assay, Knockdown, Control, Plasmid Preparation, Comparison, Marker, Over Expression

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 3 MKRN1 and SNIP1 interaction. A Prediction of protein interaction with MKRN1 using the STRING database. B Prediction of protein interaction with MKRN1 using the IntAct database. C The experimental process of MKRN1 quantitative proteomics and ubiquitination modification omics. D Expression distribution of SNIP1 (upper) and TRA2A (lower) in colorectal tumour and normal tissues. E Univariate and multifactorial Cox analyses of P-values, hazard rate, and confidence intervals for gene expression and clinical characteristics. F, G WB analysis showing that MKRN1 expression level affects SNIP1 protein expression. H Confocal microscopy showing MKRN1 and SNIP1 localisation (Scale bar: 20 µm). I, J Forward and reverse validation of MKRN1 interactions with SNIP1 in HCT116 and HCT15 cells using co-immunoprecipitation (Co-IP) and WB. K Validation of exogenous MKRN1 interaction with SNIP1 in HCT15 cells using Co-IP and WB. *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Quantitative Proteomics, Ubiquitin Proteomics, Modification, Expressing, Gene Expression, Confocal Microscopy, Biomarker Discovery, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 5 MKRN1 induces EMT in CRC cells by degrading SNIP1 protein. A, B WB was used to determine the transfection rate of SNIP1 in HCT116 and HCT15 cells. C, D WB was used to measure expression levels of epithelial and mesenchymal markers following SNIP1 knockdown and overexpression. E, F Transwell assays for migration and invasion of SNIP1 knockdown and overexpressing cells (Scale bar: 50 µm). G WB was used to determine the level of major EMT proteins in HCT116 cells co-transfected with control, sh1-MKRN1, and sh-SNIP1. H WB analysis of the level of major EMT proteins in HCT15 cells co-transfected with vector, OE-MKRN1, and OE-SNIP1. I Transwell assay of the migration ability of HCT116 cells after co-transfection with control, sh1-MKRN1, and sh-SNIP1 (Scale bar: 50 µm). J Transwell assay of HCT15 cells co-transfected with vector, OE-MKRN1, and OE-SNIP1 for cell migration (scale bar: 50 µm). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: Transfection, Expressing, Knockdown, Over Expression, Migration, Control, Plasmid Preparation, Transwell Assay, Cotransfection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation.

doi: 10.1186/s13046-023-02788-w

Figure Lengend Snippet: Fig. 7 MKRN1 promotes tumour proliferation and metastasis in vivo. A Comparative graph showing the number of intestinal lesions in the MKRN1 [+ / +] and MKRN1 [f/f] groups. B Haematoxylin–eosin (H&E) staining of the intestine of both groups of mice (scale bar: 100 μm). C H&E staining of the liver in the two groups of mice (scale bar: 100 µm; scale bar: 20 µm). D IHC staining for E-cadherin, MKRN1, SNIP1, and TGF-β1 in the intestinal tissues of the two groups of mice (scale bar: 100 µm). E Western blotting analysis of E-cadherin, MKRN1, SNIP1, and TGF-β1 protein expression in intestinal tissues of the two groups of mice. F MKRN1 facilitates the TGF-β signalling via ubiquitination and degradation of SNIP1, thereby promoting EMT in CRC cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Mutant Adenomatous polyposis coli (Apc) MKRN1 conditional knockout mice were purchased from Cyagen Biologicals, Ltd., and were categorized into groups as follows: Apc [MU/ +], Mkrn1 [+ / + , pVillin-Cre], Apc [MU/ +], and Mkrn1 [flox/flox, pVillin-Cre].

Techniques: In Vivo, Staining, Immunohistochemistry, Western Blot, Expressing, Ubiquitin Proteomics

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) Unbiased TF target enrichment analysis of RNA-seq data from A375 cells overexpressing SLNCR . (B) DATNA of TCGA RNA-seq samples of patients with melanoma. (C) Kaplan-Meier plots for patients with high levels of SLNCR and high levels of E2F1 (top left), high SLNCR and low E2F1 (bottom left), low SLNCR and high E2F1 (top right), and low SLNCR and low E2F1 (bottom right) identified using the TCGA SKCM STAR normalized dataset. Log rank p values ( p ) are indicated. (D) Matrigel invasion assays of A375 cells transfected with SLNCR overexpression or empty control plasmid and infected with scramble or E2F1-knockdown (KD) short hairpin RNA (shRNA). (E) Proliferation assay of A375 transfected as in (D). (F) Matrigel invasion assays of WM1575 (left) and WM1976 (right) infected with scramble or E2F1-KD shRNA. (G) Proliferation assay of WM1575 (left) and WM1976 (right) infected as in (F). In (A)–(G), **** p < 0.0001. For (D)–(G), data are represented as mean ± SD. Scale bar represents 100 μm. See also and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: RNA Sequencing, Transfection, Over Expression, Control, Plasmid Preparation, Infection, Knockdown, shRNA, Proliferation Assay

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) Schematic presentation of SLNCR fragments assayed for E2F1 binding by pairwise Y3H. Predicted E2F1 BSs are marked with red triangles. (B) Predicted E2F1 BSs mapped onto SLNCR secondary structure. (C) REMSA with biotin-labeled BS1–5 RNA oligonucleotides as single-strand, double-strand, or hybrid fragments, incubated with purified E2F1. (D) REMSA using biotin-labeled BS1–5 (double-strand), BS1+2, BS2+3, and BS4+5 RNA oligonucleotides with E2F1 with and without competition by unlabeled versions of the same RNA oligonucleotides. For (C) and (D), gray lines indicate separate gels, and black lines were added for better visualization. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Binding Assay, Labeling, Incubation, Purification

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA of recombinant E2F1 incubated with biotin-labeled 42-mer dsDNA (left) or 60-mer BS2+3 RNA (right) probes. The labeled 42-mer dsDNA was competed by titrating increasing amounts of unlabeled 60-mer BS2+3 RNA, and vice versa, the labeled 60-mer BS2+3 RNA was competed by titrating increasing amounts of unlabeled 42-mer dsDNA. In each experiment, the third lane from the left has 10 μM unlabeled oligonucleotide of the same species as the labeled nucleotide to show specific binding. (B) REMSA of recombinant E2F1 DBD incubated with biotin-labeled 42-mer dsDNA (left) or 60-mer BS2+3 SLNCR RNA (right) probes. The labeled 42-mer dsDNA was competed by titrating increasing amounts of unlabeled BS2+3 RNA, and vice versa, the labeled 60-mer BS2+3 RNA was competed by titrating increasing amounts of unlabeled 42-mer dsDNA. In each experiment, the third lane from left has 18μM unlabeled DNA binding site and 5 μM of unlabeled 60-mer BS2+3 RNA to show specific binding. (C) Representative structure models of E2F1 DBD +60-mer RNA from 5 MD runs. (D) Representative structure models of E2F1 DBD +42-mer dsDNA from 3 MD runs. (E) Averaged residue interaction energies of E2F1 DBD with the 15-mer reference dsDNA (gray), the 42-mer DNA 5′ motif (light blue), the 42-mer dsDNA 3′ motif (dark blue), and 60-mer RNA (red) calculated by MMGBSA. Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. (F) Number of wild-type (WT) and mutant E2F1 DBD interactions at the base, phosphate, and sugar moieties of 15-mer dsDNA, 42-mer dsDNA 5′ and 3′ motifs, and 60-mer BS2+3 RNA. See also and , , , , , , , and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Recombinant, Incubation, Labeling, Binding Assay, Residue, Mutagenesis

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A–C) Amount of protein contacts per nucleotide calculated by MMGBSA at base, phosphate, or sugar moieties. (A) Calculated protein contacts for the E2F1 DBD +15-mer dsDNA model. The top graph shows contacts with the sense strand (5′ to 3′), and the bottom graph shows protein contacts with the antisense strand (3′ to 5′). The E2F1 BS is shaded. (B) Calculated protein contacts for the E2F1 DBD +42-mer dsDNA model. The top graph shows contacts with the sense strand (5′ to 3′), and the bottom graph shows protein contacts with the antisense strand (3′ to 5′). The 5′ and 3′ motifs are shaded. (C) Calculated protein contacts for the E2F1 DBD +60-mer BS2+3 RNA model. Base-pairing interactions as predicted by RNAFold are shown as dot-and-bracket annotations below the sequence. BS2 and BS3 are shaded. (D) Table summarizing the protein contacts with base, phosphate, and sugar moieties in each MD model. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Sequencing

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA of γ-P-labeled 42-mer dsDNA (left) or biotin-labeled 60-mer BS2+3 RNA, (right) incubated with WT or mutant (R166H or L132E) E2F1 DBD or full-length E2F1 and DP1. (B and C) Averaged residue interaction energies calculated by MMGBSA of WT and mutant E2F1 DBD with 15-mer reference dsDNA (B) or 60-mer BS2+3 RNA (C). Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. WT systems are first presented in and are re-plotted here for comparison with the mutants. See also .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Labeling, Incubation, Mutagenesis, Residue, Comparison

Journal: Cell reports

Article Title: LncRNA SLNCR phenocopies the E2F1 DNA binding site to promote melanoma progression

doi: 10.1016/j.celrep.2025.115608

Figure Lengend Snippet: (A) REMSA using biotin-labeled 60-mer BS2+3 RNA incubated with recombinant E2F1 or DP1 or both with increasing amounts of DP1. (B) Representative structure from 3 independent simulations of E2F1 DBD +DP1 DBD +15-mer dsDNA showing the arrangement of the ternary system. E2F1 (salmon), DP1 (gray), and 15-mer reference dsDNA (blue). (C) Model structure of the E2F1 DBD +60-mer SLNCR RNA with DP1 docked to fit the interaction interface of E2F1 (salmon), DP1 (gray), and RNA (orange). (D) Averaged residue interaction energies of DP1 DBD calculated using MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsDNA and E2F1 DBD +DP1 DBD +15-mer dsRNA models. The RNA in the latter system was created by directly converting 15-mer dsDNA (by adding O2′) to get a 15-mer dsRNA backbone. Gray dashed line indicates −9.8 kcal/mol threshold for strong interactions. (E) Amount of protein contacts per nucleotide calculated by MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsDNA model at base, phosphate, or sugar moieties contributed by E2F1 DBD (left) and those contributed by DP1 DBD (right). The top graphs show contacts with the sense strand (5′ to 3′), and the bottom graphs show protein contacts with the anti-sense strand (3′ to 5′). The E2F1 BS is shaded. (F) Amount of protein contacts per nucleotide calculated by MMGBSA of the E2F1 DBD +DP1 DBD +15-mer dsRNA model as described in (E). See also and and .

Article Snippet: Protein production plasmids were created by introducing synthetic gene fragments containing wildtype, L132E and R166H mutant E2F1 DBD (residues 110–194) with an N-terminal His6-tag followed by a TEV protease cleavage site into pET28a SpyCatcherSnoopCatcher (gifted from Mark Howarth, Addgene plasmid #72324) by Gibson assembly.

Techniques: Labeling, Incubation, Recombinant, Residue

Journal: REPRODUCTION

Article Title: KIF1-binding protein interacts with KIF3A in haploid male germ cells

doi: 10.1530/rep-15-0173

Figure Lengend Snippet: Figure 4 KBP interacts with KIF3A in the mouse testis. (A) KIF3A was co-immunoprecipitated from the mouse testis lysate with anti-KBP antibody. A specific KIF3A band was also visible in the total testis lysate (input) and in the supernatant after immunoprecipitation (SN). Immunoprecipitation from the Kif3a KO testis lysate (KO) was used as a negative control. IgG shows the equal amount of IgG heavy chain in the immunoprecipitations from both WTand KO lysates. (B) KBPand KIF3A colocalized in the cytoplasm of late round spermatids in the mouse testis cryosections. (C) KBP colocalized with KIF3A in the manchette of elongating spermatids. (D) ptKBP colocalization with a-tubulin in the manchette. Scale barZ10 mm.

Article Snippet: Generation of germ-cell specific Kif3a knockout mice Kif3a fl/wt mice were bought from the Mutant Mouse Resource and Research Centers (MMRRC) (The Jackson Laboratory, Bar Harbor, ME, USA) and the mice expressing transgenic Cre under the Ngn3 promoter were kindly provided by Dr P L Herrera (University of Geneva, Switzerland.)

Techniques: Immunoprecipitation, Negative Control

Journal: REPRODUCTION

Article Title: KIF1-binding protein interacts with KIF3A in haploid male germ cells

doi: 10.1530/rep-15-0173

Figure Lengend Snippet: Figure 5 Localization of KBP in the late CBs is not affected in the Kif3A KO testis. Colocalization of KBP with TSKS on drying-down preparations of Kif3a KO elongating spermatids resembled the localization in the WT. However, the late CB appeared fragmented in steps 11–14 elongating spermatids. Scale barZ10 mm.

Article Snippet: Generation of germ-cell specific Kif3a knockout mice Kif3a fl/wt mice were bought from the Mutant Mouse Resource and Research Centers (MMRRC) (The Jackson Laboratory, Bar Harbor, ME, USA) and the mice expressing transgenic Cre under the Ngn3 promoter were kindly provided by Dr P L Herrera (University of Geneva, Switzerland.)

Techniques:

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A ) Schematic representation of clathrin-coated pit induction. The system, triggered by rapamycin, consists of a membrane anchor (mCherry-FRB fused to a membrane targeting domain) and a clathrin hook (clathrin-binding protein fused to FKBP and GFP). Targeting the anchor to mitochondria using Tom70p in MitoTrap. ( B ) Stills from live cell imaging of a HeLa cell expressing FKBP-β2-GFP (green), and the anchor, MitoTrap (red), treated with 200 nM rapamycin as indicated (orange bar). Scale bar, 2 µm. See . ( C ) Representative confocal micrographs of HeLa cells before (light orange bar) and 2 min after 200 nM rapamycin treatment (dark orange bar). Cells expressing MitoTrap (red) with either our standard clathrin hook (FKBP-β2-GFP), clathrin-binding deficient mutant (FKBP-β2-Y815A/∆CBM-GFP), or GFP-FKBP. In B and C, mitochondria were also labeled with MitoTracker Deep Red (blue) and orange arrowheads indicate MitoPits, where present. Inset, ×5 zoom. Scale bar, 10 µm. ( D ) Typical confocal micrograph of cells expressing FKBP-β2-GFP (green) and MitoTrap (red), treated with rapamycin (200 nM), fixed and stained with anti-PDHE2/E3 (blue). Inset, ×3 zoom. Scale bar, 5 µm. ( E ) Analysis of the spatial organization of MitoPits. ( i ) Line profile through the MitoPit shown in D, aligned to the FKBP-β2-GFP peak at 0 µm. Each of three channels is shown. (ii) Spatially averaged line profiles, aligned to the FKBP-β2-GFP peak at 0 µm, mean ± standard deviation (SD) is shown. (iii) Width of profiles for each channel in the dataset. (iv) Relative distance from the peak of FKBP-β2-GFP to the peak of MitoTrap (red) or PDHE2/3 (blue) for each profile in the dataset. Box plots indicate median, IQR, 9th and 91st percentiles. Each dot represents a profile.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Membrane, Binding Assay, Live Cell Imaging, Expressing, Mutagenesis, Labeling, Staining, Standard Deviation

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells coexpressing the indicated anchor protein together with FKBP-β2-GFP, FKBP-β2(Y815A/∆CBM)-GFP, or GFP-FKBP and treated with 200 nM rapamycin. The following anchors were used: ( A ) FRB-mCherry-Sec61β (ER), ( B ) FRB-mCherry-Giantin(3131–3259) (Golgi), and ( C ) Lamp1-mCherry-FRB (lysosomes), Insets, ×5 zoom. Scale bar, 10 µm.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques:

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A–D ) Representative confocal micrographs of MitoPit-derived vesicle formation under different approaches to inhibit dynamin or Drp1 function. ( A ) Dominant-negative dynamin-1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either Dyn1 WT-mCherry WT or Dyn1 K44A-mCherry (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). ( B ) Chemical inhibition: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green), treated with control compound (Dynole 31–2, 30 µM) or Dynole 34–2 (30 µM) for 25 min and rapamycin (200 nM) for the final 10 min. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( C ) Dynamin triple knockout: inducible dynamin TKO mouse embryonic fibroblasts expressing dark MitoTrap and FKBP-β2-GFP (green), treated with vehicle (Control) or 3 µM Tamoxifen (TKO) for 2 days prior to transfection and with rapamycin (200 nM, 10 min) for clathrin-coated pit (CCP) induction. Fluorescent human transferrin (Tfn 647, blue) indicates endocytic activity, mitochondrial matrix was stained with anti-PDHE2/E3 (red). ( D ) Dominant-negative Drp1: HeLa cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either mCherry-Drp1 WT or mCherry-Drp1 K38A (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). Insets, ×5 zoom. Scale bars, 10 µm. ( E–H ) SuperPlots showing the percentage of free spots for each condition. Colors represent replicates, dots represent cells, solid dots represent the mean of each replicate. Indicated p values from Dunnett’s post hoc test ( E, H ) or Student’s t -test ( F, G ).

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Derivative Assay, Dominant Negative Mutation, Expressing, Staining, Inhibition, Control, Activity Assay, Triple Knockout, Transfection

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: ( A ) Representative confocal micrographs of MitoPit-derived vesicle formation in cells expressing dark MitoTrap and FKBP-β2-GFP (green) alone (–) or in combination with either mCherry-Vps4a WT or E228Q mutant (red), treated with rapamycin (200 nM, 30 min), stained with anti-PDHE2/E3 (blue). ( C ) Representative confocal micrographs of MitoPit-derived vesicle formation in cells expressing dark MitoTrap and FKBP-β2-GFP (green) pre-treated with Latrunculin B (1 µM) or control as indicated. Cells were treated with rapamycin (200 nM, 30 min), and stained with Acti-stain 555 (red) and anti-PDHE2/E3 (blue). Insets, ×5 zoom. Scale bars, 10 µm. ( B, D ) SuperPlots showing the percentage of free spots for each condition in A and C. Colors represent replicates, dots represent cells, solid dots represent the mean of each replicate. Indicated p values from Student’s t-test.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Derivative Assay, Expressing, Mutagenesis, Staining, Control

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells expressing dark MitoTrap and mCherry- Epsin-2 ( A ) or mCherry-Fcho2 ( E ) (red) with either clathrin hook (FKBP-β2-GFP) or GFP-FKBP (green). Cells were treated with 200 nM rapamycin before staining for PDHE2/E3 (blue). Inset, ×5 zoom. Scale bars, 10 μm. Box plots to compare colocalization ( B, F ) and spot density ( C, G ). Each dot represents a cell. Box plots indicate median, IQR, 9th and 91st percentiles. Spots of GFP and mCherry were detected and quantified. Colocalization is shown as the percentage of GFP spots that coincided with Epsin-2 or Fcho2 spots (left), or the percentage of Epsin-2 or Fcho2 spots that coincided with GFP spots (right). ( D, H ) Waffle plots to visualize the median number of spots per 100 μm 2 that were positive for GFP only (green), clathrin only (red), or both (yellow).

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Expressing, Staining

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet: Representative confocal micrographs of HeLa cells coexpressing a red fluorescent protein-tagged accessory protein together with dark MitoTrap and either FKBP-β2-GFP or GFP-FKBP control. Cells were treated with 200 nM rapamycin and immunostained with anti-PDHE2/E3/A647. Accessory proteins were sigma subunits of the AP1, AP2, and AP3 complexes (σ1-mCherry, σ2-mCherry, or σ3-mCherry, left) or mCherry-amphiphysin 1, Endophilin-FL-RFP, Hip1R-tDimer-RFP, or mCherry-SNX9, as indicated. Insets, ×5 zoom. Scale bars, 10 µm.

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Control

Journal: eLife

Article Title: Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

doi: 10.7554/eLife.78929

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Dyn1 K44A-mCherry , This paper , - , See Materials and methods - Molecular Biology section. Modification of Addgene plasmid #34681. GFP replaced by mCherry. Available from the corresponding author..

Techniques: Triple Knockout, Recombinant, Modification, Plasmid Preparation, Mutagenesis

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) HEK293T cells were transfected with p53, Isg15-modifying enzymes and difference oncogenes (Src, Ras, Myc). In some cases, proteosome inhibitor MG132 (25 μM) was added. After Ni-beads pulldown, Isg15-modified p53 was analyzed by Western blotting with 1801 antibody. (B) Different oncogenes increase the interaction between p53 and Herc5. HEK293T cells were transfected with HA-p53, Flag-Herc5 and difference oncogenes (Src, Ras, Myc). Flag-Herc5 was immunoprecipitated using anti-Flag M2-conjugated beads, and p53 in the complexes was detected by Western blotting. (C) Isg15 siRNA knockdown enhances Src-mediated p53 stabilization. HEK293T cells were transfected with HA-p53, Src, and control or Isg15 siRNAs. Cell lysates were analysed by Western blotting using indicated antibodies. (D) Overexpression of Ras or Myc increases Src phosphorylation as a read-out of its activity. HEK293T cells were transfected with Ras or Myc. The Src phosphorylation was analyzed by Western blotting using phospho-Src Tyr416 antibody.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Transfection, Modification, Western Blot, Immunoprecipitation, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockdown by siRNA enhances Src activity-mediated p53 stabilization. The Src mutant (8A7F, R388A/Y527F) was stably transfected in HCT116 cells with Flag-tagged endogenous p53. The cells were transfected with control or Isg15 siRNA. 48h after transfection, the cells were treated with imidazole (5 mM) to activate Src activity. Cytosolic (C) and Nuclear (N) fractions were then separated, and the level of p53 was analyzed by Western blotting. Parp and RhoGDI were used as nuclear and cytosolic markers, respectively. (B) The increase of Src activity by chemical rescue promotes p53 tyrosine phosphorylation. The Src mutant (8A7F, R388A/Y527F) was stably transfected in HCT116 cells with Flag-tagged endogenous p53. After treatment of MG132 (25 μM) for 1.5h, the cells were treated with imidazole (5 mM) to activate Src activity, and collected at the indicated times. Cytosolic (C) and Nuclear (N) fractions were then separated. p53 was immunoprecipitated using anti-flag M2 beads and analyzed with phospho-Tyrosine antibody. Parp and RhoGDI were used as nuclear and cytosolic markers, respectively.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Activity Assay, Mutagenesis, Stable Transfection, Transfection, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Src phosphorylates p53 in vitro . In vitro kinase assay was performed by incubating purified His-tagged p53 with Src. The products were analysed by Western blotting using anti-phospho-Tyrosine antibody. (B) Src increases Tysosine phosphorylation of p53 in HEK293T cells. His-p53 was co-transfected Src and analyzed by Western blotting with 1801 antibody after Ni-beads pulldown. (C) Src phosphorylates p53 at Tyr126 and Tyr220. HEK293T cells were transfected with Src and WT, Y126F, Y220F, or 2F (Y126F+Y220F) His-53. p53 was precipitated with Ni-beads and analyzed with phospho-Tyrosine antibody. The results were quantified by densitometry and analyzed by GelPro software (lower panel). (D) Phospho-mimicking mutations ofTyr126 and Tyr220 increases p53 ISGyaltion. HEK293T cells transfected with WT, Y126D, Y220D, or 2D (Y126D+Y220D) mutants of p53 were analyzed for p53 ISGylation after Ni-beads pull-down. (E) Phospho-mimicking mutations of Tyr126 and Tyr220 increase p53 interaction with Herc5. HEK293T cells were transfected with WT, Y126D, Y220D, or 2D (Y126D+Y220D) p53 mutants and Flag-Herc5. Flag-Herc5 was immunoprecipitated and p53 was analyzed by Western blotting and results were quantified by densitometry (lower panel). (F) Y220C mutation increases p53 ISGylation. HEK293T cells transfected with WT, Y220C, or Y220D p53 mutants together with Isg15-modifying enzymes were analyzed for p53 ISGylation after Ni-beads pull-down.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: In Vitro, Kinase Assay, Purification, Western Blot, Transfection, Software, Immunoprecipitation, Mutagenesis

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockout increases unfolding and folding form of p53 in the transformed cells. Lysates from V-Src transformed mouse embryo fibroblasts (MEFs) (WT or Isg15 knockout) were immunoprecipitated with p53 antibodies Ab1620 or Ab240. The immunoprecipitated p53 was analysed by Western blotting. (B) Knockout of Isg15 increases the expression of p21 gene in transformed cells. RT-PCR was performed to analyse the p21 expression of V-Src transformed WT or Isg15 knockout MEFs cells. (C&D) Isg15 knockout enhances p53-mediated inhibition of transformation. (C) V-Src transformed WT, Isg15 knockout, or Isg15/p53 double knockout MEFs were grown in soft agar. Colonies were stained with MTT and counted 3 weeks later. (D) Transformed MEFs were injected into NSG nude mice. Tumors were collected and analyzed 21d after injection.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Knock-Out, Transformation Assay, Immunoprecipitation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition, Double Knockout, Staining, Injection

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Isg15 knockout impairs K-ras-induced lung tumors. 10-week-old Kras mice (control mice, n=10 (including 5 WT and 5 Isg15 Het mice); Isg15 knockout mice, n=8) were sacrificed, and lung lesions were counted (shown with arrows). (B) Isg15 knockout decrease the proliferation of K-ras-induced lung cancer. The lung collected from Isg15 Het or Isg15 knockout K-ras mice was sectioned and stained with Ki67 antibody. The positive cells of Ki67 in tumor region were showed and quantified. (C) Knockout of Isg15 increases the expression of p21 gene in K-ras-induced lung cancer. The K-ras-induced tumors were dissected from lungs containing cancer lesions. RT-PCR was performed to analyse the p21 mRNA expression. (D) Knockdown of Isg15 enhances the Nutlin-mediated inhibition of cell proliferation. MTT assay were performed with MCF7 cells transfected with either control, Isg15 siRNA, p53 siRNA or a combination of siRNAs in the absence or presence of Nutlin (3 μM). The difference of proliferating activity between 24h to 72h after seeding was shown in graphs.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Knock-Out, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Inhibition, MTT Assay, Transfection, Activity Assay

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: (A) Knockdown of Isg15 increases DNA damage-induced p53 response. HCT116 cells were transfected with control or Isg15 siRNAs, or together with p53 siRNA. Cells were irradiated with 8 Gy of IR (upper panel) or treated with UV (30 J/m 2 ) (lower panel) and collected at the time points indicated. Cell lysates were analysed by Western blotting using indicated antibodies. (B) Knockdown of Isg15 increases DNA damage-induced inhibition of survival. HCT116 cells that transfected as described in (A) were processed in colony formation assay after treatment with 1Gy of IR. 3000 cells were plated for each condition. (C) Isg15 knockout increases unfolding and folding form of p53 in the Isg15 knockdown cells. HCT116 cells were transfected with control or Isg15 siRNAs, then were irradiated with 8 Gy of IR for 6h. The cell lysates were collected and immunoprecipitated with p53 antibodies Ab1620 or Ab240. For testing the specificity of the conformation-specific antibody, the lysates were incubated at 37°C for 8 min to denature folded p53.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Transfection, Irradiation, Western Blot, Inhibition, Colony Assay, Knock-Out, Immunoprecipitation, Incubation

Journal: Oncotarget

Article Title: Oncogene-mediated regulation of p53 ISGylation and functions

doi:

Figure Lengend Snippet: In normal cells, ISGylation primarily targets misfolded dominant-negative form of p53 thus promoting a total p53 activity (1). In cancer cells, the level of Isg15 is increased and the presence of different oncogenes promotes the interaction between p53 and Herc5 to increase the p53 ISGlyation. As a result, native p53 is targeted by Isg15-dependent degradation reducing the overall p53 activity.

Article Snippet: Antibodies used were the following: PAb1801 (sc98; Santa Cruz), p53 rabbit antibody (no. 9282; Cell Signaling ISG15 (AP1150a; ABGENT), phosphotyrosine (610012; BD Biosciences), p21 (OP79; Oncogene Science), actin (A2066; Sigma), HA (16B12; Covance), His (sc8036; SantaCruz), GST (RPN1236; Amersham), FLAG M2 antibody (Sigma), and FLAG rabbit antibody (no.2368, Cell Signaling).

Techniques: Dominant Negative Mutation, Activity Assay