ruvbl2  (Cell Signaling Technology Inc)

Journal: FEBS Open Bio

Article Title: Characterization of WAC interactions with R2TP and TTT chaperone complexes linking glucose and glutamine availability to mTORC1 activity

doi: 10.1002/2211-5463.70085

Figure Lengend Snippet: WAC interacts with the R2TP complex. (A) Pull‐down experiment testing the interaction between WAC‐3xFlag and the R2TP complex. WAC‐3xFlag, used as bait, was incubated with a purified, already assembled R2TP complex. (B) To map the interaction of WAC with the subunits of the R2TP complex, a similar experiment as in (A) was performed to test the interaction with the RUVBL1‐RUVBL2 complex. Similar experiments are shown but using RPAP3 (C) and RPAP3‐PIH1D1 (D) as prey. Assays were performed as explained in the Methods section, similarly to the experiments shown in Fig. .

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti‐FLAG M2 antibody (Sigma‐Aldrich, F1804, 1 : 2000), HA (Abcam #1091591; 1 : 1000), mLST8_GBL 86B8 (Cell Signaling #3274; 1 : 1000), mTOR (7C10, Cell signaling #2972; 1 : 1000), RagA/B D8B5 (Cell Signaling #4357; 1 : 1000), RAPTOR (24C12) (Cell signaling, #2280; 1 : 1000), S6 ribosomal protein (Cell Signaling #2217; 1 : 1000), p‐S6 ribosomal protein (Ser240/244) (Cell Signaling #2215; 1 : 1000), TELO2 (15975‐1‐AP, Proteintech, Rosemont, IL, USA; 1 : 1000), TTI1 (A303‐451A, Bionova Scientific; 1 : 1000), TTI2 (A303‐476A, Bionova Scientific, Fremont, CA, USA; 1 : 500), RPAP3 (Invitrogen #PA5‐58335; 1 : 500), RUVBL1 (Cell signaling #12300; 1 : 500), RUVBL2 (Cell signaling #8959; 1 : 500), PIH1D1 (Invitrogen PA5‐61482, 1 : 500).

Techniques: Incubation, Purification

Journal: FEBS Open Bio

Article Title: Characterization of WAC interactions with R2TP and TTT chaperone complexes linking glucose and glutamine availability to mTORC1 activity

doi: 10.1002/2211-5463.70085

Figure Lengend Snippet: Analysis of gene and protein levels for WAC, mTORC1, R2TP, and TTT across cancer types. Gene (A) and protein (B) levels of the subunits of the mTOR‐WAC‐RUVBL1‐RUVBL2‐TTT complex in 10 cancer types with CPTAC tumor samples from LinkedOmics. Z‐scores are shown to represent normalized expression levels, with red indicating relatively high expression and blue indicating relatively low expression. The number of cancer types showing a positive correlation in gene (C) and protein (D) levels between subunits of the mTOR‐WAC‐RUVBL1‐RUVBL2‐TTT complex across 10 cancer types. The number within each box indicates the number of cancer types with a positive correlation out of the 10 tested. A positive correlation is defined as a Pearson correlation coefficient > 0.2 with a P ‐value < 0.01. (E) Distribution of WAC protein expression in tumor samples versus matched normal samples across cancer types (where normal samples are available). Statistically significant differences between tumor and normal conditions were assessed using the two‐sided Wilcoxon rank sum test. Units in the Y axis correspond to protein expression levels in LinkedOmics, provided as log₂‐transformed MS1 intensities, which represent relative protein abundances measured via mass spectrometry and normalized for comparative analyses. (F) Differences in protein levels of the components of the WAC‐RUVBL1‐RUVBL2‐TTT complex between tumor and adjacent normal tissue (NAT) across cancer types. Red indicates higher expression in tumors compared to normal samples, while blue indicates higher expression in normal samples compared to tumors. Statistically significant differences were assessed using the two‐sided Wilcoxon rank sum test. The meta P ‐value represents pan‐cancer significance when all samples are analyzed together using the two‐sided Wilcoxon rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001. BRCA, breast invasive carcinoma; CCRCC, clear cell renal cell carcinoma; COAD, colon adenocarcinoma; GBM, glioblastoma; HNSCC, head and neck squamous cell carcinoma; LSCC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; OV, ovarian serous cystadenocarcinoma; PDAC, pancreatic ductal adenocarcinoma; UCEC, uterine corpus endometrial carcinoma.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti‐FLAG M2 antibody (Sigma‐Aldrich, F1804, 1 : 2000), HA (Abcam #1091591; 1 : 1000), mLST8_GBL 86B8 (Cell Signaling #3274; 1 : 1000), mTOR (7C10, Cell signaling #2972; 1 : 1000), RagA/B D8B5 (Cell Signaling #4357; 1 : 1000), RAPTOR (24C12) (Cell signaling, #2280; 1 : 1000), S6 ribosomal protein (Cell Signaling #2217; 1 : 1000), p‐S6 ribosomal protein (Ser240/244) (Cell Signaling #2215; 1 : 1000), TELO2 (15975‐1‐AP, Proteintech, Rosemont, IL, USA; 1 : 1000), TTI1 (A303‐451A, Bionova Scientific; 1 : 1000), TTI2 (A303‐476A, Bionova Scientific, Fremont, CA, USA; 1 : 500), RPAP3 (Invitrogen #PA5‐58335; 1 : 500), RUVBL1 (Cell signaling #12300; 1 : 500), RUVBL2 (Cell signaling #8959; 1 : 500), PIH1D1 (Invitrogen PA5‐61482, 1 : 500).

Techniques: Expressing, Transformation Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Testosterone-Induced Metabolic Changes in Seminal Vesicle Epithelial cells Alter Plasma Components to Enhance Sperm Fertility

doi: 10.1101/2024.01.16.575926

Figure Lengend Snippet: (A) Tracing of oxygen consumption rate (OCR) basal and injection with 10 nM oleic acid (OA) or OA + 40 nM Etomoxir (Eto). (B) The sperm, after 1 hour of incubation with or without seminal vesicle secretions (SV) from nontreated (Ctrl) or flutamide-treated mice(Flu), were used for IVF, and the cleavaged oocytes were observed. The absolute number of oocytes collected from the oviduct and the cleavage rate are shown. (C) Western blot analysis for phosphotyrosine (P-Tyr) in capacitated spermatozoa (Ctrl; 1-hour incubation in HTF medium) and treated with SV from healthy mice or 10 nM oleic acid (OA). (D) Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. (E) Flow cytometric analysis of the sperm after 1 hour incubation in control medium (HTF) and medium containing SV or 10 nM OA, using fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; to distinguish between acrosome-reacted and non-reacted cells) and propidium iodide (PI; to distinguish between dead and viable cells). (F) The percentage of viable sperm with an acrosome reaction (PI-, PNA-FITC+) was evaluated by flow cytometry in the 4th quadrant (4Q; red square). Data are mean ± SEM. At least three independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. (B) Dunnett’s test was used to analyze the cleavage rate. Different letters represent significantly different groups. (D,F) Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies: anti-phosphotyrosine antibody (1:10,000; 8959; Cell Signaling), anti-GLUT4 antibody (1:100; ab33780; Abcam), anti-ACLY antibody (1:10,000; ab40793; Abcam) or anti-α/β-Tubulin antibody (1:1,000; 2148S; Cell Signaling).

Techniques: Injection, Incubation, Western Blot, Control, Flow Cytometry, Transformation Assay