Journal: FEBS Open Bio

Article Title: Human CKAP 2 L shows a cell cycle‐dependent expression pattern and exhibits microtubule‐stabilizing properties

doi: 10.1002/2211-5463.13864

Figure Lengend Snippet: Cell cycle‐dependent changes in CKAP2L transcript and protein levels in human foreskin fibroblasts. Normal human foreskin fibroblasts were arrested at the G 1 ‐S boundary using aphidicolin; released; and harvested at the indicated time points. (A) The cells were analyzed by RT‐PCR to determine the relative abundance of human CKAP2L mRNA. CKAP2 mRNA was detected as a comparison. GAPDH served as a loading control. (B) The cells were analyzed by western blot to determine the relative abundance of human CKAP2L, CKAP2, cyclin B1, and phospho‐histone H3 at Ser10 (pHH3) protein. α‐tubulin served as a loading control. (C) The samples from the indicated time points were analyzed by flow cytometry to monitor the cell cycle progression. See Fig. for the axial values. (D) The time‐ and cell cycle‐dependent changes in CKAP2L protein level and the indicated proteins. the intensities of protein bands shown in Panel B were quantified using Science Lab 2001 Image Gauge Ver. 4.0 and normalized to those of α‐tubulin bands. For each protein, the highest band intensity was set as the reference (i.e., 1.0), and the relative intensities of bands (for each protein) are shown here. For the ease of comparison between the levels of CKAP2L and the indicated protein, the CKAP2L curve was reproduced for each panel.

Article Snippet: Mouse monoclonal antibodies against α‐tubulin (clone B‐5‐1‐2) and cyclin B1 (clone GNS1) were purchased from Sigma and Santa Cruz (Dallas, TX, USA), respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Comparison, Control, Western Blot, Flow Cytometry

Journal: FEBS Open Bio

Article Title: Human CKAP 2 L shows a cell cycle‐dependent expression pattern and exhibits microtubule‐stabilizing properties

doi: 10.1002/2211-5463.13864

Figure Lengend Snippet: Expression of CKAP2L in human and mouse tissues and human cell lines. (A) The transcript levels of human CKAP2L , along with CKAP2 , CCNB1 , and TOP2A , were measured by RT‐PCR in various human organs and tissues. ACTB served as a loading control. (B) The transcript levels of mouse Ckap2l and Ckap2 were measured by RT‐PCR in various mouse organs and tissues. Gapdh served as a loading control. (C) The protein levels of human CKAP2L and CKAP2 were measured in the indicated human cell lines of different tissue origins by western blot. α‐tubulin served as a loading control.

Article Snippet: Mouse monoclonal antibodies against α‐tubulin (clone B‐5‐1‐2) and cyclin B1 (clone GNS1) were purchased from Sigma and Santa Cruz (Dallas, TX, USA), respectively.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: FEBS Open Bio

Article Title: Human CKAP 2 L shows a cell cycle‐dependent expression pattern and exhibits microtubule‐stabilizing properties

doi: 10.1002/2211-5463.13864

Figure Lengend Snippet: Subcellular localization of endogenous CKAP2L and GFP‐CKAP2L during mitosis. (A) HEK293 cells were co‐immunostained with a rabbit polyclonal anti‐human CKAP2L antibody (Alexa488; green) and a monoclonal antibody against α‐tubulin (Cy3; red). DAPI was used to stain nuclei (blue). Panels show representative images of cells at different phases of mitosis showing subcellular localization of endogenous CKAP2L. In cells undergoing cytokinesis, two foci of CKAP2L staining (arrows) were observed. (B) HEK293 cells were transfected with a GFP‐CKAP2L expression construct. On the following day, the cells were fixed and stained for α‐tubulin (Cy3; red) and nuclei (DAPI; blue). Localization of GFP‐CKAP2L signal during different phases of mitosis is shown in green and resembled that of endogenous CKAP2L. Scale bars 10 μm.

Article Snippet: Mouse monoclonal antibodies against α‐tubulin (clone B‐5‐1‐2) and cyclin B1 (clone GNS1) were purchased from Sigma and Santa Cruz (Dallas, TX, USA), respectively.

Techniques: Staining, Transfection, Expressing, Construct

Journal: FEBS Open Bio

Article Title: Human CKAP 2 L shows a cell cycle‐dependent expression pattern and exhibits microtubule‐stabilizing properties

doi: 10.1002/2211-5463.13864

Figure Lengend Snippet: Endogenous CKAP2L localizes to the nucleus during interphase. (A) HEK293 cells were transfected with a GFP‐CKAP2L (green) expression construct; fixed; and immunostained for α‐tubulin (Cy3; red) and nuclei (DAPI; blue). The panel shows representative images of GFP‐CKAP2L‐expressing, non‐mitotic HEK293 cells in interphase. GFP‐CKAP2L localized to both microtubules and nucleus or to microtubules alone (arrow). (B) Immunostaining of endogenous CKAP2L (Alexa488; green) and α‐tubulin (Cy3; red) shows that some interphase cells exhibit nuclear staining that co‐localizes with DAPI (blue). (C) Western blot showing total cell lysate (Total) or subcellular fractions (Nuclear vs. Cytosolic) of asynchronous HEK293 cells. Lamin A/C was detected to validate the nuclear fraction, and α‐tubulin served largely as a cytosolic marker. Immunoblot for endogenous CKAP2L protein shows that the majority of the protein is found in the nuclear fraction in asynchronously cycling HEK293 cells. Scale bars 10 μm.

Article Snippet: Mouse monoclonal antibodies against α‐tubulin (clone B‐5‐1‐2) and cyclin B1 (clone GNS1) were purchased from Sigma and Santa Cruz (Dallas, TX, USA), respectively.

Techniques: Transfection, Expressing, Construct, Immunostaining, Staining, Western Blot, Marker

Journal: FEBS Open Bio

Article Title: Human CKAP 2 L shows a cell cycle‐dependent expression pattern and exhibits microtubule‐stabilizing properties

doi: 10.1002/2211-5463.13864

Figure Lengend Snippet: Overexpression of GFP‐CKAP2L increases the stability of microtubules and results in a delay in mitotic progression. (A) HEK293 cells transfected with a GFP‐CKAP2L (green) construct were stained for α‐tubulin (Cy3; red) and nuclei (DAPI; blue). Representative images show ‘microtubule bundling’ (arrows) among GFP‐CKAP2L‐expressing cells. (B) HEK293 cells transfected with a GFP‐CKAP2L (green) construct were immunostained for acetylated α‐tubulin (Cy3; red), an indicator of stable microtubules. Cells transfected with a GFP vector served as a control. GFP or GFP‐CKAP2L‐transfected cells (green) are indicated by arrows. Neighboring, untransfected cells are indicated by asterisks. Strong acetylated α‐tubulin staining (Cy3; red) is only observed in GFP‐CKAP2L‐expressing cells. (C) Time‐lapse microscopy of GFP‐CKAP2L‐expressing HEK293 cells. The upper panels show phase‐contrast images, and the bottom panels show GFP‐CKAP2L fluorescent images. The GFP (control)‐expressing cell (upper panels) undergo mitosis within an hour, while the GFP‐CKAP2L‐expressing cell (bottom panels) takes more than 7 h to complete mitosis. The numbers on top show time lapsed in hours:minutes before and after the initiation of mitosis which was set as 0:00. Scale bars 10 μm.

Article Snippet: Mouse monoclonal antibodies against α‐tubulin (clone B‐5‐1‐2) and cyclin B1 (clone GNS1) were purchased from Sigma and Santa Cruz (Dallas, TX, USA), respectively.

Techniques: Over Expression, Transfection, Construct, Staining, Expressing, Plasmid Preparation, Control, Time-lapse Microscopy