Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

doi: 10.1074/jbc.M114.591545

Figure Lengend Snippet: Phosphorylation of TPX2 at Thr72 is inhibited by the Cdk inhibitor roscovitine. A, the levels of Thr(P)72 were reduced by roscovitine treatment. HeLa cells were first treated with 100 ng/ml of nocodazole for 16 h and then once synchronized, treated with dimethyl sulfoxide (as a control), or 20 and 40 μm roscovitine for 30 min. Cells were harvested, lysed, and TPX2 was immunoprecipitated with pan-TPX2 Abs (clone 184). SDS-PAGE was performed and followed by Western blotting with Thr(P)72 and pan-TPX2 Abs (clone 184). B, cyclin B1 levels were also used to confirm that roscovitine-treated cells had remained in mitosis. The levels of p-Cdk/MAPK substrates (PX(S*/T*)P or (S*/T*)PX(R/K) motif) were also used to confirm the effectiveness of the treatment. Actin levels were used as loading control.

Article Snippet: Antibodies Primary Abs against Cdk1, Cdk2 (Santa Cruz), Thr(P) 72 TPX2 (homemade, described above), Cyclin B (Abcam), α-actin (Chemicon), TPX2 (clone 183, epitope: 150–200 amino acids of human TPX2, Novus Biologicals; clone 184, epitope: 700–749 amino acids of TPX2, Novus Biologicals), phospho-Cdk/MAPK substrates (34B2, Cell Signaling), Cy3 conjugated-β-tubulin (Sigma), p-Aurora A (ab18318, Abcam), and monoclonal TPX2 (18D5-1, Abcam) were used for Western blots, immunoprecipitations, and/or immunofluorescence experiments.

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Developmental biology

Article Title: Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

doi: 10.1016/j.ydbio.2017.08.025

Figure Lengend Snippet: Fig. 2. LBX1 is phosphorylated at S223, S227, and S234. (a) Myc-taged Lbx1 and points mutants were transiently transfected to NIH3T3 cells. At 24 h post transfection, cells were treated with 100 nM PMA for 30 min and lysed. Wild type (Wt) LBX1 and mutants are detected by Western blotting using anti-Myc-tag and IRdye800 conjugated anti-mouse IgG. Phosphorylation of wild type (Wt) LBX1 was upregulated by PMA and quantitated in (b). Phosphorylation of S227A was also up- regulated by PMA treatment, suggesting LBX1 is phosphorylated at multiple sites. (c) S223 and S234 were predicted to be ERK substrates and an anti-PXSP antibody was used to detect the phosphorylation at these sites. Myc-taged Lbx1 and points mutants were transiently transfected to NIH3T3 cells. At 24 h post transfection, cells were treated with 100 nM PMA for 30 min and lysed. Wild type (Wt) LBX1 and mutants are detected by Western blotting using anti-Myc and anti-Erk substrate (PXSP) antibody. IRdye800 conjugated anti-mouse IgG and IRdye700 conjugated anti-Rabbit IgG antibodies were used as secondary antibodies. Phosphorylation at S234 but not S223 was detected by anti-PXSP antibody, revealing that S234 alone is a likely to be an ERK substrate. Mobility difference between LBX1S227A and LBX1S223A S227A double mutant reveals that S223A is also phosphorylated. Note that the phosphorylations at S223 and S227 were both increased by PMA treatment.

Article Snippet: For in-vitro experiments the following antibodies and reagents were used: anti-Myc mouse IgG2a monoclonal antibody (9b11, Cell Signalling Technology; 1/2000), anti-PXSP rabbit IgG monoclonal antibody (34B2, Cell Signalling Technology; 1/1000), anti-Flag rabbit IgG monoclonal antibody (f7425, Sigma; 1/1000) Alexa Fluor 488 conjugated anti-mouse antibody (ab150113, Abcam; 1:400), IRDye700 and 800 secondary antibodies were purchased from LI-COR and diluted to 1:15000.

Techniques: Transfection, Western Blot, Phospho-proteomics, Mutagenesis