cub02b control beads  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: RNF13 mediates pH- and Ca 2+ -dependent regulation of lysosomal positioning

doi: 10.1101/2025.03.19.644102

Figure Lengend Snippet: (A) Western blot analysis of endogenous ARL8B in HeLa cells transfected with either an empty vector (EV) or plasmids encoding RNF13-HA or RNF13 C243S (CS)-HA at increasing concentrations. The arrow indicates the N-glycosylated form of RNF13. Protein levels were normalized to β-actin. Relative protein levels are shown as mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test. (ns, not significant; *p < 0.05; **p < 0.01). (B) Western blot analysis of indicated proteins in HeLa cells treated with DMSO (-), MG132 (25 μM), or NH 4 Cl (20 mM) for 6 h of: (left panel) normal cells; (right panel) cells co-transfected with ARL8B- HA and either an EV or plasmid encoding RNF13-FLAG. Increase in p62 levels confirmed the effectiveness of MG132 and NH 4 Cl. (C) Western blot analysis for the ubiquitination assay of ARL8B. (Left panel) HeLa cells were transfected with plasmids encoding ARL8B-FLAG and T7-UB, along with either an EV, RNF13-HA, or RNF13 CS- HA plasmids. Cell lysates were subjected to immunoprecipitation (IP) using FLAG antibodies, followed by Western blotting. (Right panel) Lysates from cells transfected with EV, RNF13-FLAG, or RNF13 CS- FLAG were incubated with ubiquitin-affinity or control beads. Beads were collected, and the eluates were analyzed by Western blotting. For all ubiquitination assays, cells were pretreated with MG132 (25 μM) for 6 h before harvesting. Western blots were performed using the indicated antibodies. (D) Western blot analysis of endogenous ARL8B levels following starvation. Cell extracts from MCF7 cells stably expressing control shRNA (shGFP) or RNF13 shRNA (shRNF13) were immunoblotted after incubation with either complete, EBSS, or serum-free media for 2 h. Relative ARL8B levels, normalized to β-actin, were determined and presented as mean ± SD (n = 3). Statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparisons test’s test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (E) (Left panel) Immunofluorescence analysis of LAMP1 in the samples in (D) at 2 h. Samples were immunostained with LAMP1 for lysosomes, CD147 for cell boundary, and DAPI for nucleus. Scale bar, 10 μm. (Right panel) A SuperPlot shows the ratio of perinuclear LAMP1 to total LAMP1, determined through shell analysis (see the STAR Methods). For each condition, three independent experiments were performed. Over 20 cells were analyzed in individual experiment, which is distinguished by different colors. Small circles represent individual data points, while large circles denote the mean of each experiment. Horizontal lines represent the mean ± SD of the means from three independent experiments. Statistical significance was assessed by two-way ANOVA with Tukey’s test (ns, not significant; *p < 0.05; ***p < 0.001). (F) Western blot analysis of endogenous ARL8B in MCF7 cells stably expressing control shRNA (shGFP) or RNF13 shRNA (shRNF13) after incubation with alkaline media for 2 h. The relative ARL8B levels, normalized to β-actin, were determined and presented as mean ± SD (n=3). Statistical significance was analysed by two-way ANOVA with Tukey’s test (ns, not significant; *p < 0.05; **p < 0.01). (G) Western blot analysis for ubiquitination assay of endogenous ARL8B. HeLa cells were treated as indicated for 2 h and cell lysates incubated with either control or ubiquitin affinity beads as described in panel (C). The whole cell lysates and the eluates were analyzed by western blotting with indicated antibodies. (H) (Left panel) Immunofluorescence analysis of LAMP1 in the samples shown in (F) at 2 h. Samples were immunostained with LAMP1 for lysosomes, CD147 for cell boundary, and DAPI for nucleus. Scale bar, 10 μm. (Right panel) A SuperPlot illustrates the ratio of perinuclear LAMP1 to total LAMP1 as described in panel (E). Horizontal lines indicate the mean ± SD of the means from three independent experiments. Statistical significance was evaluated using two-way ANOVA with Tukey’s test (ns, not significant; *p < 0.05; ***p < 0.001).

Article Snippet: Ubiquitination of endogenous ARL8B was analyzed using the SignalSeeker Ubiquitination Detection Kit (Cytoskeleton) following the manufacture’s instruction.

Techniques: Western Blot, Transfection, Plasmid Preparation, Comparison, Ubiquitin Assay, Immunoprecipitation, Incubation, Control, Stable Transfection, Expressing, shRNA, Immunofluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: The long non-coding RNA NEAT1 contributes to aberrant STAT3 signaling in pancreatic cancer and is regulated by a metalloprotease-disintegrin ADAM8/miR-181a-5p axis

doi: 10.1007/s13402-024-01001-0

Figure Lengend Snippet: NEAT1 regulates STAT3 signaling in an ADAM8-dependent manner. a siRNA-mediated knockdown of NEAT1 in Panc89 cells and western blot analysis of STAT3 ubiquitination. The levels of total STAT3, pSTAT3, and GAPDH, which were used as loading controls, are shown below. b Similar to ( a ), NEAT1 was knocked down in Panc1_A8 cells, which increased the level of ubiquitinated STAT3, concomitant with decreased levels of total STAT3 and pSTAT3. GAPDH served as a loading control. c Detection of ubiquitinated STAT3 levels in Panc89_WT, Panc89_A8KO and botezomib treated Panc89_A8KO cells by Western blot. Notably, the ubiquitinated STAT3 levels in Panc89_WT and botezomib treated Panc89_A8KO cells were higher than those in Panc89_A8KO cells; compared with those in Panc89_A8KO cells, the total STAT3 and pSTAT3 levels in the Panc89_WT and Panc89_A8KO cells treated with bortezomib (1 ng/mL) for 16 hours increased. d Compared with those in Panc1_WT cells, ADAM8 overexpression in Panc1 cells or treatment with bortezomib (1 ng/mL) for 16 hours decreased the levels of ubiquitinated STAT3 while increased total STAT3 and pSTAT3 levels. e As a result of NEAT1 knockdown in Panc89_WT cells, the expression of potential target genes of STAT3, such as ICAM1, was analyzed by RT-qPCR. f NEAT1 knockdown inhibited ICAM1 mRNA expression in Panc1_A8 cells. g ADAM8 knockout inhibited ICAM1 mRNA expression in Panc89_A8KO cells, and treatment with bortezomib (1 ng/mL) for 16 hours restored ICAM1 mRNA expression in Panc89_A8KO cells. h ADAM8 overexpression promoted ICAM1 mRNA expression in Panc1 cells, and treatment with bortezomib (1 ng/mL) for 16 hours restored ICAM1 mRNA expression in Panc1 cells. Data are presented as mean ± S.D and as statistical test, Mann-Whitney and Kruskal-Wallis tests were used with * p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Ubiquitination assays were conducted using a Signal Seeker™ Ubiquitination Detection Kit (Cytoskeleton Inc., Denver, CO, USA).

Techniques: Knockdown, Western Blot, Control, Over Expression, Expressing, Quantitative RT-PCR, Knock-Out, MANN-WHITNEY

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: The long non-coding RNA NEAT1 contributes to aberrant STAT3 signaling in pancreatic cancer and is regulated by a metalloprotease-disintegrin ADAM8/miR-181a-5p axis

doi: 10.1007/s13402-024-01001-0

Figure Lengend Snippet: Graphical sketch of how ADAM8 affects STAT3 levels via the regulation of miR-181a-5p and NEAT1. In the left half, either wild-type or protease-deficient full-length ADAM8 can suppress miR-181a-5p expression in PDAC cells, leading to high expression levels of NEAT1 in the cytoplasm. NEAT1 binds to STAT3, and this complex is not degraded in the proteasome; thus, STAT3 signaling remains high in ADAM8-expressing cells (red arrow below) to promote the downstream effects of STAT3, such as proliferation, migration, and invasion. The right half shows the situation in which ADAM8 is either deficient or the cytoplasmic domain is missing. This leads to high levels of miR-181a-5p that suppress NEAT1 expression and ubiquitination of STAT3 with subsequent degradation in the proteasome, thereby downregulating STAT3 signaling in PDAC cells. Abbreviations: A8/ADAM8, A disintegrin and metalloproteinase 8; MP, metalloprotease; DC, a disintegrin-like and a cysteine-rich; EGF, epidermal growth factor-like; TM, transmembrane; CD, cytoplasmic tail; SH3, Src homolog 3; WT, wild type; KO, knockout; EQ, the protease-inactive variant ADAM8 with amino acid exchange E to Q at aa position 335; DCD, an ADAM8 construct lacking the cytoplasmic domain; STAT3, signal transducer and activator transcription 3; p-STAT3: phosphorylated STAT3; NEAT1: nuclear enriched abundant transcript 1

Article Snippet: Ubiquitination assays were conducted using a Signal Seeker™ Ubiquitination Detection Kit (Cytoskeleton Inc., Denver, CO, USA).

Techniques: Expressing, Migration, Knock-Out, Variant Assay, Construct

Journal: Nature Communications

Article Title: Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

doi: 10.1038/s41467-024-50311-2

Figure Lengend Snippet: a , b Detection of LnaB-mediated conversion of PR-Ub into ADPR-Ub by mass spectrometric analysis. Excised protein bands from SDS-PAGE gels corresponding to PR-Ub prior to the reaction or ADPR-Ub after incubation with ATP, LnaB, and Actin were digested with trypsin and analyzed by mass spectrometry. A reference fragment T 12 ITLEVEPSDTIENVK 27 was present in both samples with similar abundance (a left panel). The abundance of the fragment with PR-modified R42 was high in the PR-Ub samples but became almost undetectable after reaction with LnaB, ATP, and Actin, which was accompanied by the increase of the ADPR-modified fragment. An MS/MS spectrum indicating ADPR modification of R42 was shown in ( b ). c A reaction scheme depicting the conversion of PR-Ub into ubiquitin by LnaB and MavL. The AMPylation activity of LnaB first converts PR-Ub into ADPR-Ub, which is further reduced into ADP-ribose and ubiquitin by MavL. The AMP moiety defined by a dashed line rectangle indicates the chemical group added to PR-Ub by LnaB. d The use of ADPR-Ub produced from PR-Ub by LnaB in protein modification by the phosphodiesterase (PDE) activity of SdeA. PR-Ub was incubated in the indicated reactions and the ability to ubiquitinate Rab33b was detected by the formation of higher MW species detected by immunoblotting with the Flag-specific antibody. Native ADPR-Ub was included as a control (1 st lane). e Conventional ubiquitination by ubiquitin produced by MavL and LnaB from PR-Ub. A series of reactions containing PR-Ub and combinations of relevant proteins were allowed to proceed for 1 h at 37 °C. The products were boiled for 5 min at 95 °C, and a cocktail containing E1, E2, SidC (E3), and ATP was added, self-ubiquitination of SidC was detected by immunoblotting with a ubiquitin-specific antibody.

Article Snippet: For the SdeA-mediated ubiquitination reaction, 2 μg His 6 -LnaB, 1.5 μg Actin (Cytoskeleton, cat# APHL99) and 6 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and 1 mM ATP for 1 h at 37 °C.

Techniques: SDS Page, Incubation, Mass Spectrometry, Modification, Tandem Mass Spectroscopy, Activity Assay, Produced, Western Blot, Control

Journal: Nature Communications

Article Title: Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

doi: 10.1038/s41467-024-50311-2

Figure Lengend Snippet: Ubiquitin is converted into ADPR-Ub by the mART activity of SidEs, which is used to modify proteins by phosphoribosyl ubiquitination. The reversal of the modification produced PR-Ub, which is converted into native ubiquitin by sequential reactions catalyzed by LnaB and MavL. Note that both ADPR-Ub and PR-Ub may interfere with canonical ubiquitin signaling and that ADPR-Ub produced from PR-Ub by LnaB may be used by the PDE activity of SidEs for protein modification.

Article Snippet: For the SdeA-mediated ubiquitination reaction, 2 μg His 6 -LnaB, 1.5 μg Actin (Cytoskeleton, cat# APHL99) and 6 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and 1 mM ATP for 1 h at 37 °C.

Techniques: Activity Assay, Modification, Produced

Journal: Nature Communications

Article Title: Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

doi: 10.1038/s41467-024-50311-2

Figure Lengend Snippet: a , b Detection of LnaB-mediated conversion of PR-Ub into ADPR-Ub by mass spectrometric analysis. Excised protein bands from SDS-PAGE gels corresponding to PR-Ub prior to the reaction or ADPR-Ub after incubation with ATP, LnaB, and Actin were digested with trypsin and analyzed by mass spectrometry. A reference fragment T 12 ITLEVEPSDTIENVK 27 was present in both samples with similar abundance (a left panel). The abundance of the fragment with PR-modified R42 was high in the PR-Ub samples but became almost undetectable after reaction with LnaB, ATP, and Actin, which was accompanied by the increase of the ADPR-modified fragment. An MS/MS spectrum indicating ADPR modification of R42 was shown in ( b ). c A reaction scheme depicting the conversion of PR-Ub into ubiquitin by LnaB and MavL. The AMPylation activity of LnaB first converts PR-Ub into ADPR-Ub, which is further reduced into ADP-ribose and ubiquitin by MavL. The AMP moiety defined by a dashed line rectangle indicates the chemical group added to PR-Ub by LnaB. d The use of ADPR-Ub produced from PR-Ub by LnaB in protein modification by the phosphodiesterase (PDE) activity of SdeA. PR-Ub was incubated in the indicated reactions and the ability to ubiquitinate Rab33b was detected by the formation of higher MW species detected by immunoblotting with the Flag-specific antibody. Native ADPR-Ub was included as a control (1 st lane). e Conventional ubiquitination by ubiquitin produced by MavL and LnaB from PR-Ub. A series of reactions containing PR-Ub and combinations of relevant proteins were allowed to proceed for 1 h at 37 °C. The products were boiled for 5 min at 95 °C, and a cocktail containing E1, E2, SidC (E3), and ATP was added, self-ubiquitination of SidC was detected by immunoblotting with a ubiquitin-specific antibody.

Article Snippet: For the SdeA-mediated ubiquitination reaction, 2 μg His 6 -LnaB, 1.5 μg Actin (Cytoskeleton, cat# APHL99) and 6 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and 1 mM ATP for 1 h at 37 °C.

Techniques: SDS Page, Incubation, Mass Spectrometry, Modification, Tandem Mass Spectroscopy, Activity Assay, Produced, Western Blot, Control

Journal: Nature Communications

Article Title: Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

doi: 10.1038/s41467-024-50311-2

Figure Lengend Snippet: Ubiquitin is converted into ADPR-Ub by the mART activity of SidEs, which is used to modify proteins by phosphoribosyl ubiquitination. The reversal of the modification produced PR-Ub, which is converted into native ubiquitin by sequential reactions catalyzed by LnaB and MavL. Note that both ADPR-Ub and PR-Ub may interfere with canonical ubiquitin signaling and that ADPR-Ub produced from PR-Ub by LnaB may be used by the PDE activity of SidEs for protein modification.

Article Snippet: For the SdeA-mediated ubiquitination reaction, 2 μg His 6 -LnaB, 1.5 μg Actin (Cytoskeleton, cat# APHL99) and 6 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 and 1 mM ATP for 1 h at 37 °C.

Techniques: Activity Assay, Modification, Produced