Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Primers used.

Article Snippet: HEK293T cells were transfected with a human QKI5 and human SEMA7A 3′UTR-reporter plasmid (SwitchGear Genomics) through the use of polyethylenimine (3 μg/1 μg DNA; Polysciences, Inc.).

Techniques:

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: RNA-immunoprecipitation showing quaking protein binding to the 3′UTR of SEMA7A. (a) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 using an IgG control or QKI5 Ab. QKI5 and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 5; * P < 0.05. (b) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 and 3′UTR of SEMA7A using an IgG control or QKI5 Ab. SEMA7A 3’UTR and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 3; * P < 0.05.

Article Snippet: HEK293T cells were transfected with a human QKI5 and human SEMA7A 3′UTR-reporter plasmid (SwitchGear Genomics) through the use of polyethylenimine (3 μg/1 μg DNA; Polysciences, Inc.).

Techniques: RNA Immunoprecipitation, Protein Binding, Over Expression, Control, Immunoprecipitation

Journal: Cell Cycle

Article Title: RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

doi: 10.1080/15384101.2016.1235103

Figure Lengend Snippet: Downregulation of QKI-5 mRNA expression in ccRCC tissues and cell lines. (A) QKI-5 mRNA expression levels in 34 paired primary ccRCC tissues (T) and adjacent noncancerous tissues (N) were determined by qPCR assays. P < 0.001, paired t test. (B) qPCR analysis of QKI-5 expression in ccRCC cell lines and HK-2 immortalized renal proximal epithelial tubular cells. (C-E) Correlation of QKI-5 gene expression with tumor T stage, M status, and grade of differentiation. chi-square test, Primary data were taken from TCGA ccRCC data set (https://tcga-data.nci.nih.gov/tcga/). The boxes represent the 25th to 75thpercentiles, and horizontal lines within the box represent median values. The whiskers represent the lowest and highest value in the 25th percentile minus 1.5IQR and 75th percentile plus 1.5IQR regions, respectively. (C)The 25thpercentiles,75thpercentiles,median in T1-2: 9.751695, 10.82361, 10.24365;T3-4: 9.427069, 10.51334, 9.962113.(D) The 25thpercentiles,75thpercentiles,median in M0: 9.651339, 10.74372, 10.19386;M1: 9.514634, 10.50172, 9.968011.(E) the 25thpercentiles,75thpercentiles,median in G1-2: 9.701859, 10.8063, 10.24195;G3-4: 9.547901, 10.6237, 10.04023.(F) Kaplan-Meier survival curve of overall survival for QKI-5 expression in 513 mLRCC patients from TCGA ccRCC dataset (https://tcga-data.nci.nih.gov/tcga/).

Article Snippet: 44,45 The primary antibodies used included those for QKI-5 (Merck, 03–112); RASA1 (Santa Cruz, SC-63); cyclin A, D1, and E; p21; p27 (all from Santa Cruz Biotechnology); and α-tubulin (Sigma, T6199), as well as a number of antibodies from Cell Signaling Technology, including those for ERK (4780), phospho-ERK1/2 (5726), p38(8690), phospho-p38 (Thr180/Tyr182, 4511), and Raf-1 (9422).

Techniques: Expressing

Journal: Cell Cycle

Article Title: RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

doi: 10.1080/15384101.2016.1235103

Figure Lengend Snippet: Overexpression of QKI-5 inhibits the proliferation of ccRCC cells in vitro and in vivo. (A–E) 786-O and A-498 cells with stably overexpressing QKI-5 or transfected with empty vector (Vec) were analyzed as follows. (A) QKI-5 mRNA expression levels were determined by qPCR assays. (B) QKI-5 protein expression levels were determined by immunoblotting; α-tubulin was used as a loading control. (C) Cell proliferation was determined by the MTS assay; (D) Colony formation ability; representative micrographs (left) and quantification (right) of crystal violet-stained cells from 3 independent experiments; (E) Control or QKI-5-overexpressing 786-O cells were inoculated subcutaneously into nude mice (n = 5/group). Tumor volumes were measured (left) and weighed (right) on the last days of the experiment. Representative images of isolated tumors (middle) are presented; *P < 0.05, **P < 0.01, student's t-test.

Article Snippet: 44,45 The primary antibodies used included those for QKI-5 (Merck, 03–112); RASA1 (Santa Cruz, SC-63); cyclin A, D1, and E; p21; p27 (all from Santa Cruz Biotechnology); and α-tubulin (Sigma, T6199), as well as a number of antibodies from Cell Signaling Technology, including those for ERK (4780), phospho-ERK1/2 (5726), p38(8690), phospho-p38 (Thr180/Tyr182, 4511), and Raf-1 (9422).

Techniques: Over Expression, In Vitro, In Vivo, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, MTS Assay, Staining, Isolation

Journal: Cell Cycle

Article Title: RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

doi: 10.1080/15384101.2016.1235103

Figure Lengend Snippet: QKI-5 directly upregulated RASA1 expression via regulating its mRNA stability. (A and B) mRNA expression levels were determined by real-time qPCR assays. The expression levels of RASA1 were significantly upregulated in 786-O and A-498 cells stably overexpressing QKI-5, whereas that of Axl, Frk, Igfbp7 and Ahr were not. *P< 0.05, Student t test. (C and D) QKI-5 and RASA1 mRNA expression was significantly downregulated in the matched primary ccRCC tissues (T) in contrast to the adjacent noncancerous tissues (N). (E) RASA1 mRNA levels were positively correlated with QKI-5 expression in 34 pairs of ccRCC tissues as determined by real-time qPCR (GAPDH was used as reference genes. Linear regression was indicated, r2 = 0.5293; P < 0.0001).(F) Immunoblotting analysis of RASA1 in control or QKI-5-overexpressing 786-O and A-498 cells, α-tubulin was used as a loading control. Data presented here are a representative of 3 different experiments. Densitometry of Western blot to analyze the relative protein expression by using Image J software (G) Direct interaction between RASA1 and QKI-5. QKI-5 overexpressing 786-O cells were immunoprecipitated with anti- QKI-5 antibody or the negative control IgG. The presence of RASA1 mRNA in the immunoprecipitation was detected by RT-PCR and visualized by ethidium bromide staining.

Article Snippet: 44,45 The primary antibodies used included those for QKI-5 (Merck, 03–112); RASA1 (Santa Cruz, SC-63); cyclin A, D1, and E; p21; p27 (all from Santa Cruz Biotechnology); and α-tubulin (Sigma, T6199), as well as a number of antibodies from Cell Signaling Technology, including those for ERK (4780), phospho-ERK1/2 (5726), p38(8690), phospho-p38 (Thr180/Tyr182, 4511), and Raf-1 (9422).

Techniques: Expressing, Stable Transfection, Western Blot, Software, Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Cell Cycle

Article Title: RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

doi: 10.1080/15384101.2016.1235103

Figure Lengend Snippet: QKI-5 inhibits the proliferation of ccRCC cells via regulating RASA1 and the RAS-MAPK signaling pathway. (A and B) QKI-5-expressing and control A-498 cells were transiently transfected with RASA1siRNA (si-rasa1-1,si-rasa1-3) or scrambled control siRNA (si-ctrl). RASA1 expression levels were evaluated by real-time qPCR assays (A) and immunoblotting analysis(B). (C) MTS analysis of cell proliferation in A-498 cells described in A. (** p,0.01, Student's t-test). (D) Immunoblotting was used to assess the regulatory effect of QKI-5 and RASA1 in the regulation of RAS signaling pathway.

Article Snippet: 44,45 The primary antibodies used included those for QKI-5 (Merck, 03–112); RASA1 (Santa Cruz, SC-63); cyclin A, D1, and E; p21; p27 (all from Santa Cruz Biotechnology); and α-tubulin (Sigma, T6199), as well as a number of antibodies from Cell Signaling Technology, including those for ERK (4780), phospho-ERK1/2 (5726), p38(8690), phospho-p38 (Thr180/Tyr182, 4511), and Raf-1 (9422).

Techniques: Expressing, Transfection, Western Blot

Journal: Cell Cycle

Article Title: RNA-binding protein QKI-5 inhibits the proliferation of clear cell renal cell carcinoma via post-transcriptional stabilization of RASA1 mRNA

doi: 10.1080/15384101.2016.1235103

Figure Lengend Snippet: QKI-5 promotes cell cycle arrest at the G1/S-phase transition through P27 and Cyclin D1, involving MAPK signaling pathway. (A and B) 786-O or A-498 cells stably overexpressing QKI-5 or transfected with empty vector (Vec) were subjected to cell cycle analysis by flow cytometry. Images and qualification of the cell cycle distribution in 3 experiments are shown; *P < 0.05, **P < 0.01, Student t test. (C) Immunoblotting analysis of cyclin A, cyclin D1, cyclin E, and their inhibitors p21 and p27 in the indicated cell lines. α-tubulin was used as a loading control. (D) Immunoblotting analysis of phosphorylated ERK, total ERK, phosphorylated p38, and total p38 in the indicated cell lines. α-tubulin was used as the loading control.

Article Snippet: 44,45 The primary antibodies used included those for QKI-5 (Merck, 03–112); RASA1 (Santa Cruz, SC-63); cyclin A, D1, and E; p21; p27 (all from Santa Cruz Biotechnology); and α-tubulin (Sigma, T6199), as well as a number of antibodies from Cell Signaling Technology, including those for ERK (4780), phospho-ERK1/2 (5726), p38(8690), phospho-p38 (Thr180/Tyr182, 4511), and Raf-1 (9422).

Techniques: Sublimation, Stable Transfection, Transfection, Plasmid Preparation, Cell Cycle Assay, Flow Cytometry, Western Blot

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/eLife.43322

Figure Lengend Snippet: Non-R-mAb antibodies used in this study. Table lists Abs used in this study outside of the R-mAbs whose generation is described here. For each Ab the name, immunogen used in Ab generation, source and RRID number in the Antibody Registry, form and concentration/dilution used, and specific use in this paper is detailed.

Article Snippet: N147/6 , Fusion protein aa 1–341 (full-length) of human QKI-5 , Mouse IgG2b mAb, NeuroMab RRID: AB_10671658 , Tissue culture supernatant, 1:5 , 4.

Techniques: Concentration Assay, Affinity Purification, Recombinant