Journal: Cell Proliferation

Article Title: Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

doi: 10.1111/j.1365-2184.2009.00582.x

Figure Lengend Snippet: Association of PKC‐ι, with Cdk7 in RWPE‐1 cells. Whole cell extracts (1 mg) from each time point were immunoprecipitated with rabbit polyclonal anti‐Cdk7 (5 µg) as described in the Materials and methods section. Column 1 contains both positive (+) and two negative controls (–). Positive control (+) is the whole cell lysates (100 µg); the first negative control (–) contains whole cell lysates (1 mg) plus rabbit IgG whole molecule (50 µl of 1 : 1 v/v) and the second negative control contains whole cell lysate (1 mg) plus rabbit IgG whole molecule (50 µl) and normal rabbit IgG serum (5 µg). Column 2 is cell lysates taken at indicated time points. Immunoprecipitates were separated by SDS‐PAGE and were Western blotted with anti‐PKC‐ι mouse monoclonal antibody. Physical association of PKC‐ι and Cdk7 were observed at 30 h time points (a). Immunoprecipitation (IP) with rabbit polyclonal Cdk7 (b) showed that Cdk2 is also coimmunoprecipitated (c). Western blotting of phospho‐Cdk7 (p‐Cdk7; T170) (d) and phospho‐Cdk2 (p‐Cdk2; T160) were also observed (e). Presence of Cdk2 was observed throughout the cell cycle (f) and β‐actin (g) shows equal loading of samples in each lane.

Article Snippet: Reagents and antibodies Primary antibodies were purchased from the following companies: PKC‐α, βΙ, δ, ɛ, γ, θ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); PKC‐ι (Transduction Laboratory, Lexington, KY, USA); PKC‐ζ (Upstate Biotechnology, Lake Placid, NY, USA); p‐Bad (ser‐112), p‐Bad (ser‐155), p‐Bad (ser‐136), Bad, PARP, cytochrome c, caspase‐7, survivin, Cdk2, Cdk7, β‐actin (Santa Cruz Biotechnology); cleaved PARP (Asp 214), Bcl‐x L , p‐Cdk2 (Ther160) (Cell Signalling Technology, Danvers, MA, USA); and phospho‐Cdk7‐T170 (Abgent, San Diego, CA, USA).

Techniques: Immunoprecipitation, Positive Control, Negative Control, SDS Page, Western Blot

Journal: Cell Proliferation

Article Title: Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

doi: 10.1111/j.1365-2184.2009.00582.x

Figure Lengend Snippet: PKC‐ι siRNA leads to apoptosis. Whole cell extracts of RWPE‐1 treated with control siRNA and PKC‐ι siRNA were prepared as described in the Materials and methods section and immunoblot analysis of PARP and cleaved PARP (Asp214), indicated cells are undergoing apoptosis via activation of caspase‐7. Immunoblotting for survivin and cytochrome c further demonstrated apoptosis in PKC‐ι siRNA treated cells. Western blot analysis of β‐actin shows that an equal amount of protein was loaded in each lane. Similar immunoblots were performed for DU‐145 cells. Activation of PARP and caspase‐7, combined with an increase in cytochrome c and decrease in survivin indicated apoptosis in DU‐145 cells. (b) A separate experiment was repeated for RWPE‐1 cells with control siRNA and PKC‐ι siRNA and whole cells extracts (150 µg) were analysed for Cdk7, p‐Cdk7 (T170), Cdk2, p‐Cdk2 (T160), and β‐actin to verify equal loading of protein. (c) Densitometry for p‐Cdk7 and p‐Cdk2 showed a significant decrease in PKC‐ι siRNA‐treated cells compared to control siRNA (P = 0.001 and 0.035, respectively).

Article Snippet: Reagents and antibodies Primary antibodies were purchased from the following companies: PKC‐α, βΙ, δ, ɛ, γ, θ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); PKC‐ι (Transduction Laboratory, Lexington, KY, USA); PKC‐ζ (Upstate Biotechnology, Lake Placid, NY, USA); p‐Bad (ser‐112), p‐Bad (ser‐155), p‐Bad (ser‐136), Bad, PARP, cytochrome c, caspase‐7, survivin, Cdk2, Cdk7, β‐actin (Santa Cruz Biotechnology); cleaved PARP (Asp 214), Bcl‐x L , p‐Cdk2 (Ther160) (Cell Signalling Technology, Danvers, MA, USA); and phospho‐Cdk7‐T170 (Abgent, San Diego, CA, USA).

Techniques: Western Blot, Activation Assay

Journal: Frontiers in Oncology

Article Title: PKM2 Interacts With the Cdk1-CyclinB Complex to Facilitate Cell Cycle Progression in Gliomas

doi: 10.3389/fonc.2022.844861

Figure Lengend Snippet: PKM2 physically interacts with the Cdk1-cyclin B complex. (A) Levels (top) and activity (bottom) of Cdk7 in control and PKM2 knock-down U87, T98G and LN319 cells 10 hrs following release from serum deprivation-induced arrest. (B) Levels of cyclin B1, PKM2, and Cdk1 in total cell lysates in control or PKM2 shRNA U87 cells measured at the times indicated following release from serum deprivation-induced arrest. (C) Levels of Cdk1, cyclin B, or PKM2 from Cdk1, cyclin B or PKM2 immunoprecipitates measured at the times indicated following release of control or PKM2 shRNA U87 cells from serum deprivation-induced arrest. Data in M lanes were derived from cells isolated by mitotic shake-off. I:B = Immuno Blot, IP = immunoprecipitate. Bottom panel: Densitometric analysis of Cdk1, cyclin B, or PKM2 levels in U87 cells from Cdk1, cyclin B or PKM2 immunoprecipitates. (D) Western blot analysis of cyclin B1 in control and PKM2 knock-down U87, T98G and LN319 cells. Densitometric analysis (bottom right panel) of western blot demonstrating levels of cyclin B1 in different experimental groups. *p<.05, n=3.

Article Snippet: For Cdk1-cyclin B activity analysis, cell lysates were diluted and pipetted into recombinant Cdc7 pre-coated wells (MBL International Corp), after which Mg2+ and ATP were added, and the amount of phosphorylated substrate was measured by binding to an anti-phospho-Cdk7 (T 376) antibody and a horseradish peroxidase-conjugated anti-mouse IgG.

Techniques: Activity Assay, Control, Knockdown, shRNA, Derivative Assay, Isolation, Western Blot