Journal:

Article Title: Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

doi:

Figure Lengend Snippet: Human cyclin K is associated with a potent CTD kinase activity. (A) Western blot analysis of human cell lysates probed with antibodies (α) to cyclin K. Sixty micrograms of protein from each of the following whole-cell extracts was probed with affinity-purified α-Kpep antibodies: U-2 OS, osteogenic sarcoma; WI-38, human diploid fibroblast; HepG2, hepatocellular carcinoma; and RPE (gift of A. Davis). (B) Immunoprecipitation of endogenous cyclin K protein from the [35S]methionine-labeled RPE cell line. Two microliters (2 μg) of affinity-purified α-Kpep antibodies or 5 μl of NRS was used to immunoprecipitate protein from lysates (500 μg). One microgram of cyclin K peptide (80-fold molar excess) was used to inhibit 2 μg of α-Kpep antibody. Asterisks correspond to bands associated with the cyclin K immunoprecipitation. (C) GST-CTD in vitro kinase activity associated with cyclin K. RPE whole-cell extracts (100 μg) were immunoprecipitated with either affinity-purified α-Kpep (lanes 1 to 6) or preimmune (P.I.) sera (lanes 7 and 8) that had been preincubated with (lanes 5 and 6) or without (lanes 1, 2, 3, 4, 7, and 8) peptide competitor, and the resultant complexes were used for in vitro kinase assays with (lanes 1, 2, 3, 5, and 7) or without (lanes 4, 6, and 8) the addition of a bacterially produced GST-CTD fusion protein. (D) Association of cyclin K with RNAP II in RPE. Immunoprecipitations were performed with 100 μg of RPE cell extract by using 20 μl each of protein A-Sepharose beads, protein A-Sepharose beads coupled with preimmune (P.I.) sera, and protein A-Sepharose beads covalently cross-linked with crude α-Kpep antibodies, with or without preincubation with cyclin K peptide competitor. Western blots were then performed with anti-RNAP II (ARNA3) antibodies (upper panel) and α-KFL antibodies (lower panel) as probes.

Article Snippet: Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-K pep antibodies, affinity-purified α-K pep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods.

Techniques: Activity Assay, Western Blot, Affinity Purification, Immunoprecipitation, Labeling, In Vitro, Produced

Journal:

Article Title: Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

doi:

Figure Lengend Snippet: Cyclin K is a CAK in vitro. (A) Cyclin K is associated with a CAK activity towards cyclin A-Cdk2. Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-Kpep antibodies, affinity-purified α-Kpep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods. IPs were incubated with 0.1 μg of bacterially expressed and purified cyclin A, HA-Cdk2, and ATP. After activation, the complexes were assayed for H1 kinase activity with [γ-32P]ATP as previously described (9), and reaction products were electrophoresed by SDS-PAGE (10% polyacrylamide) and visualized by autoradiography. (B) The cyclin K-associated CAK activity requires T161 of Cdk2. CAK assays were performed as described for panel A with IPs from RPE cell extracts, except that instead of HA-Cdk2, the T161A mutant of HA-Cdk2 was used.

Article Snippet: Immunoprecipitations were performed from RPE and WI-38 cell lysates by using 2 μg each of NRS, affinity-purified α-K pep antibodies, affinity-purified α-K pep antibodies preincubated with peptide competitor, and anti-Cdk7 (Santa Cruz Biotechnology, Inc.) antibodies as described in Materials and Methods.

Techniques: In Vitro, Activity Assay, Affinity Purification, Incubation, Purification, Activation Assay, SDS Page, Autoradiography, Mutagenesis

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effect of transforming growth factor‐β1 (TGF‐β1) on expression of angiomodulin (AGM), fibronectin (FN) and α‐smooth muscle actin (α‐SMA) in two kinds of cultured human fibroblasts. Human natal dermal fibroblasts (HDFs) (a) and WI38 cells (b) were incubated with the indicated concentrations (ng/mL) of TGF‐β1 in serum‐free medium for 2 days. From each culture, the conditioned medium and cell lysates were prepared, as described in Materials and Methods. AGM and fibronectin were analyzed with the conditioned media, while α‐SMA and β‐actin as an internal loading control were done with the cell lysates. The results were reproduced in at least three separate experiments.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Cell Culture, Incubation, Control

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effects of angiomodulin (AGM) and transforming growth factor‐β1 (TGF‐β1) on growth of human fibroblasts. (a,b) Human natal dermal fibroblasts (HDFs) were incubated with the indicated concentrations of TGF‐β1 for 5 days (a) or AGM for 4 days (β) in DMEM/F12+5% FCS medium on 24‐well plates. Each point represents the mean ± SD of the numbers of cells in triplicate wells. (c) Time course of HDF growth in presence (●) or absence (○) of 10 μg/mL AGM. (d) Effect of varied concentrations of AGM on the growth of HDFs was examined in the presence (●) or absence (○) of 10 μM Smad inhibitor SB431542 (Smad inh.) for 6 days on a 96‐well plate. The cell growth was measured by the crystal violet staining. Each point represents the mean ± SD in triplicate wells. (e,f) Effects of varied concentrations of TGF‐β1 (e) or ΑGΜ (f) on the growth of WI38 cells were examined for 5 days as described in (a) and (b). Other experimental conditions are described in Materials and Methods.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Incubation, Staining