gfp tagged proteins  (ATCC)

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

doi: 10.1186/s12964-025-02628-8

Figure Lengend Snippet: Nexilin interacts with Rab7b and regulates the size of Rab7b-positive LE/Lys. A Cell lysates from PC3 and U2OS cells were analysed through Western blot using antibodies against Rab7b and tubulin as loading control. B U2OS cells transiently transfected with GFP or GFP-nexilin were lysed and subjected to immunoprecipitation with GFP magnetic agarose beads. The total lysates and immunoprecipitated proteins were analysed by Western blot using the indicated antibodies. C Bacterially expressed and purified His-Rab7a, His-Rab7b, and His-Rab9 were incubated with HeLa cell lysates. Proteins were pulled down using cobalt-coated magnetic beads and subjected to Western blot analysis using the indicated antibodies. D U2OS cells transiently transfected with GFP-Rab7b (green) were fixed and stained with rhodamine phalloidin (grey) and an antibody against nexilin (magenta) and imaged using a super-resolution confocal microscope. White square indicates the magnified areas, arrows point to areas of colocalisation. Scale bar: 5µm. E Fluorescence intensity profile of Rab7b (green), nexilin (magenta) and actin (black), measured along the indicated lines and normalised to the max fluorescence value in their respective channels. F U2OS cells were treated with control siRNA or different siRNAs against nexilin and transiently transfected with GFP-Rab7b before live cell imaging. Scale bar: 5 µm. G Quantification of the number of Rab7b-positive LE/Lys with an area larger than 3 µm 2 per cell. Data represents the mean ± s.d. from three independent experiments (n = 60 cells in total per condition), two-tailed, unpaired Student’s t -test was applied for statistical analysis. H U2OS cells were co-transfected with mCherry-Rab7b (green), and either GFP-nexilin wild type (wt), GFP-nexilin S442A (phosphorylation-mimic mutant), or GFP-nexilin S442A (phosphorylation-null mutant), shown in magenta. Scale bar: 5 µm. I Quantification of number of Rab7b-positive LE/Lys with area over 3 μm 2 per cell in cells co-transfected with mCherry-Rab7b and either GFP-nexilin wild type (wt), GFP-nexilin S442A, or GFP-nexilin S442A. Data represents the mean ± s.d. from three independent experiments (n = 30 cells in total per condition), one-way ANOVA, followed by Tukey’s post-hoc test was applied for statistical analysis. ns non-significant, * p < 0.05

Article Snippet: GFP-TRAP ® _MA for immunoprecipitation of GFP fusion proteins (Chromotek) was used according to manufacturer’s protocol.

Techniques: Western Blot, Control, Transfection, Immunoprecipitation, Purification, Incubation, Magnetic Beads, Staining, Microscopy, Fluorescence, Live Cell Imaging, Two Tailed Test, Phospho-proteomics, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Nexilin promotes calcium-dependent endo-lysosomal fission required for retrograde transport

doi: 10.1186/s12964-025-02628-8

Figure Lengend Snippet: Nexilin regulates LE/Lys size through the interaction with the lysosomal Ca 2+ channel TRPML1. A U2OS cells transiently transfected with either GFP or GFP-Nexilin were subjected to immunoprecipitation with GFP magnetic agarose beads. The total lysates and immunoprecipitated proteins were analysed through Western blot using the indicated antibodies. B U2OS cells depleted for nexilin by siRNA transfection were lysed and analysed by Western blot using the indicated antibodies. C Quantification of the levels of phosphorylated myosin light chain on Ser 19 (pMLC). The intensity of the bands from the Western blots were normalised to the total myosin light chain (MLC) levels, using tubulin as loading control. Data represents the mean ± s.d. from three independent experiments. Two-tailed, unpaired Student’s t -test was applied for statistical analysis. D U2OS cells were transiently transfected with either GFP or GFP-nexilin and subjected to immunoprecipitation with GFP magnetic agarose beads. The total lysates and immunoprecipitated proteins were analysed through Western blot using the indicated antibodies. E U2OS cells treated with either control siRNA or siRNA targeting nexilin were transiently transfected with either GFP or GFP-Rab7b. The lysates were subjected to immunoprecipitation with GFP magnetic agarose beads and analysed through Western blot, using the indicated antibodies. F Quantification of the levels of myosin II, TRPML1, or nexilin in the indicated samples immunoprecipitated using GFP magnetic agarose beads. Intensity of the bands from Western blots were normalised to the immunoprecipitated GFP-Rab7b levels, and plotted relative to the control condition. Data represents the mean ± s.d. from three independent experiments. Two-tailed, unpaired Student’s t -test was applied for statistical analysis. G U2OS cells were treated with control siRNA or siRNAs targeting nexilin and transiently co-transfected with mCherry-Rab7b and GFP-TRPML1 before live cell imaging. Blue and white arrows points to smaller and larger endosomes (under or over 3 µm 2 in area), respectively. Scale bar: 5 μm. The graphs represent colocalisation analysis of: H Rab7b with TRPML1, I TRPML1 with Rab7b, analysed by calculating Mander’s coefficient in ImageJ. Data represents the mean ± s.d. for three independent experiments ( n = 45 cells in total per condition). Two-tailed, unpaired student’s t -test was applied for statistical analysis. J The graph displays distribution of TRPML1 mean intensity on large (area > 3 µm 2 ) and small (area < 3 µm 2 ) LE/Lys, normalised to the total mean intensity of TRPML1 per cell, upon nexilin knock down. Two-tailed, unpaired Student’s t -test was applied for statistical analysis. K U2OS cells were treated with control siRNA or siRNAs targeting nexilin and transiently transfected with GFP-Rab7b before live cell imaging. The same cells were then treated with 20 µM of the TRPML1 agonist ML-SA1 for 1 h before live cell imaging. Scale bar: 5 μm. L Quantification of the number of Rab7b-positive endosomes larger than 3 µm 2 in control cells and cells knocked down for nexilin, with and without ML-SA1 treatment. Data represents mean ± s.d. from three independent experiments ( n ≥ 58 cells in total per condition). Two-way ANOVA, followed by Šidák post-hoc test was applied for statistical analysis. ns non-significant, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: GFP-TRAP ® _MA for immunoprecipitation of GFP fusion proteins (Chromotek) was used according to manufacturer’s protocol.

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Two Tailed Test, Live Cell Imaging, Knockdown

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a , b The half-life of EN2 mRNA were determined by qRT-PCR in LINC00973 knockdown or overexpressed Cal27 and HN6 cells with time-course Actinomycin D exposure. c Experimental design of AGO2-RIP was depicted by schematic diagram (left panel). Endogenous LINC00973 was assessed by qRT-PCR in cell lyses immunoprecipitated with either AGO2 antibody or mouse IgG (right panel). d , e The putative miRNA candidates with high EN2 mRNA binding affinity were computed by TargetScan webtool ( https://www.targetscan.org/vert_80/ ), while LINC00973 sponged miRNAs (top10) were predicted by DIANA tool kits ( http://diana.imis.athena-innovation.gr/DianaTools/index.php ). Six overlapped miRNA candidates were identified ( d ). Only miR-6756-3p expression were shown with significant upregulation followed by LINC00973 silencing in both Cal27 and HN6 cells ( e ). f The expression of miR-6756-3p in 50 HNSCC clinical samples and paired normal counterparts were determined by qRT-PCR. Paired Wilcoxon rank-sum test. g Kaplan-Meier analyses of OS and DSS stratified by median miR-6756-3p expression in 50 in-house samples Log-rank test. h , i Schematic diagram depicted the experimental design of MS2-RIP assays to detect the direct binding between LINC00973 and miR-6756-3p in HNSCC ( h ). The LINC00973-MS2-GFP complex was pulled down by anti-GFP antibody and the interacted miR-6756-3p were assessed by qRT-PCR in Cal27, HN6, and HEK293T cells ( i ). j The sequence alignment displayed the binding sequence between miR-6756-3p and LINC00973 [LINC00973 (WT)] or 3’UTR of EN2 [3’UTR EN2 (WT)]. The miR-6756-3p binding site mutated LINC00973 and EN2 transcripts [LINC00973 (MT) or 3’UTR EN2 (MT)] were showed below. k , l Cal27 and HN6 cells transfected with LINC00973/3’UTR EN2 wild type or miR-6756-3p binding site mutated pmirGLO luciferase reporter vectors and treated with miR-6756-3p or control mimics. The luciferase activated was measured. m The expression levels of EN2 were measured by qRT-PCR in Cal27 and HN6 treated with sh-LINC00973-1 lentivirus or in combination with miR-6756-3p inhibitor (inhibitor). n The correlations between EN2, LINC00973, and miR-6756-3p expression in HNSCC clinical samples were estimated by using Spearman’s correlation. o Schematic illustration of the regulatory model of LINC00973 decoys miR-6756-3p to stabilize EN2 mRNA. Data were presented as mean ± SD, # P ≥ 0.05, * P < 0.05, ** P < 0.01, Student’s t test.

Article Snippet: Immunoprecipitation was performed using an anti-GFP antibody (0.5 μg per sample, 50430-2-AP, Proteintech, China) to pull down the MS2-GFP fusion protein together with interacted LINC00973-MS2-12× transcripts and miRNAs.

Techniques: Quantitative RT-PCR, Knockdown, Immunoprecipitation, Binding Assay, Expressing, Sequencing, Transfection, Luciferase, Control