Journal: Molecules and Cells

Article Title: Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

doi: 10.14348/molcells.2018.2266

Figure Lengend Snippet: As denoted in , IMR-90 cells were synchronized with 2 mM double thymidine arrest, incubated with 5 μM cytochalasin D or the solvent DMSO as a control at 5.5–6 h after the second release, and collected at each indicated time point after the second release. Cell lysates were resolved by 8% SDS-PAGE and blotted. Blots were probed with (A) p-ERK1/2 and p-RSK1 (Ser 380) and re-probed with anti-ERK1/2 and anti-RSK1 to observe the total amount of each protein, (B) p-ERK1/2 and p-Cdc25C (Ser 216), and re-probed with anti-ERK1/2 and anti-Cdc25C. (A, B) Cell cycle progression at G2/M was monitored by detecting p-Cdc2 (Tyr 15) followed by re-probing with anti-Cdc2 to detect the total amount of Cdc2. (C) The same samples from (A) and (B) were blotted with p-Wee1 (Ser 642) and re-probed with anti-Wee1. Each blot was re-probed with anti-actin as a loading control.

Article Snippet: Rabbit polyclonal p-Cdc2 (Tyr 15) (ab47594, Abcam, USA), mouse monoclonal anti-cyclin A (4656, Cell Signaling Technology, USA), rabbit polyclonal p-Cdc25C (Ser 216) (9528, Cell Signaling Technology), rabbit monoclonal anti-Cdc25C (ab32444, Abcam), rabbit polyclonal anti-β-actin (4967, Cell Signaling Technology), rabbit monoclonal p-Wee1 (Ser 642) (4910, Cell Signaling Technology), rabbit polyclonal anti-Wee1 (4936, Cell Signaling Technology), rabbit monoclonal anti-FLAG (F7425, Sigma), rabbit polyclonal p-ERK1/2 (Thr 202/Tyr 204) (9101, Cell Signaling), rabbit polyclonal anti-ERK1/2 (9102, Cell Signaling), rabbit monoclonal p-RSK1 (Ser 380) (ab32203, Abcam), rabbit polyclonal anti-RSK1 (9333, Cell Signaling), rabbit monoclonal histone H3 (4499, Cell Signaling Technology), and mouse monoclonal p-H3 (Ser 10) (9706, Cell Signaling Technology) were used in this study.

Techniques: Incubation, SDS Page

Journal: Molecules and Cells

Article Title: Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

doi: 10.14348/molcells.2018.2266

Figure Lengend Snippet: (A) The layout shows how the experiment was performed. IMR-90 cells were synchronized with 2 mM thymidine and transfected with DN-RSK or empty vector as described in the Materials and Methods. Cells were treated with 5 μM CD or only DMSO as a control at 5.5–6 h after the second release and collected at each indicated time point after the second release. Each cell lysate was resolved by 8% SDS-PAGE and blotted with (B) anti-FLAG and anti-actin, (C) p-ERK1/2 and p-Cdc2 (Tyr 15), (D) p-Cdc25C (Ser 216), and p-Wee1 (Ser 642). Each membrane was reprobed with anti-actin as a loading control.

Article Snippet: Rabbit polyclonal p-Cdc2 (Tyr 15) (ab47594, Abcam, USA), mouse monoclonal anti-cyclin A (4656, Cell Signaling Technology, USA), rabbit polyclonal p-Cdc25C (Ser 216) (9528, Cell Signaling Technology), rabbit monoclonal anti-Cdc25C (ab32444, Abcam), rabbit polyclonal anti-β-actin (4967, Cell Signaling Technology), rabbit monoclonal p-Wee1 (Ser 642) (4910, Cell Signaling Technology), rabbit polyclonal anti-Wee1 (4936, Cell Signaling Technology), rabbit monoclonal anti-FLAG (F7425, Sigma), rabbit polyclonal p-ERK1/2 (Thr 202/Tyr 204) (9101, Cell Signaling), rabbit polyclonal anti-ERK1/2 (9102, Cell Signaling), rabbit monoclonal p-RSK1 (Ser 380) (ab32203, Abcam), rabbit polyclonal anti-RSK1 (9333, Cell Signaling), rabbit monoclonal histone H3 (4499, Cell Signaling Technology), and mouse monoclonal p-H3 (Ser 10) (9706, Cell Signaling Technology) were used in this study.

Techniques: Transfection, Plasmid Preparation, SDS Page