Journal: International Journal of Molecular Sciences

Article Title: Modulation of Primary Cilia by Alvocidib Inhibition of CILK1

doi: 10.3390/ijms23158121

Figure Lengend Snippet: Alvocidib effect on CILK1 phosphorylation of KIF3A in cells. ( A ) GST-CILK1 or Flag-CILK1 was co-transfected with HA-KIF3A (substrate) into HEK293T cells. Forty-eight hours after transfection, cells were treated with increasing concentrations of Alvocidib in DMSO for 15 min before lysis. HA-KIF3A was immunoprecipitated from cell extracts with mouse monoclonal HA-tag antibody (12CA5). Both cell extracts and anti-HA immunoprecipitates were Western blotted for total and phospho-T672 KIF3A. ( B ) HEK293T cells co-transfected with GST-CILK1 and HA-KIF3A were treated with various doses of Alvocidib or AT7519. Cell extracts were Western blotted for total and phospho-T672 KIF3A and GST-CILK1. ( C ) The phospho-T672 versus total KIF3A ratio was plotted against the concentrations of drugs. Shown is average ± SD, n = 3 independent experiments; * p < 0.05; ** p < 0.01. ( D ) GST-CILK1 or Flag-CILK1 was co-transfected with HA-KIF3A into HEK293T cells. Cells were treated with 100 nM Alvocidib in a time course. Cell extracts were Western blotted for total and phospho-T672 KIF3A. From cell extracts, GST-CILK1 and Flag-CILK1 proteins were recovered by glutathione agarose beads and anti-FLAG M2 agarose beads, respectively, and then Western blotted by a CILK1 antibody. ( E ) HEK293T cells co-transfected with GST-CILK1 and HA-KIF3A were treated with Alvocidib in the nanomolar range for 15 min before lysis, protein extraction, and immunoprecipitation. Both cell extracts and anti-HA immunoprecipitates were Western blotted for total and phospho-T672 KIF3A. ( F ) Phospho-T672 of HA-KIF3A in anti-HA immunoprecipitates was plotted against various concentrations of Alvocidib. Shown is average ± SD, n = 3 independent experiments; ** p < 0.01.

Article Snippet: GST-tag (B-14) mouse monoclonal (sc-138) and HA-tag (12CA5) mouse monoclonal (sc-57592) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Transfection, Lysis, Immunoprecipitation, Western Blot, Protein Extraction

Journal: FEBS letters

Article Title: Overexpression of Mos(rat) proto-oncogene product enhances the positive autoregulatory loop of MyoD.

doi: 10.1016/s0014-5793(98)01192-2

Figure Lengend Snippet: Fig. 4. Mos co-expressed with MyoD activates expression of the endogenous MyoD protein. 10T1/2 cells were co-transfected with 0.5 Wg of pEMSV-E12 (lanes 1^14), 0.2, 0.5 and 1 Wg of pCMVHA-MosWT (lanes 2^4 and 9^11) or pCMVHA-MosKM (lanes 5^7 and 12^14) alone or in combination with 0.5 Wg of pCMVHa-MyoD (lanes 8^14). Forty-eight hours following transfection, cells maintained in DMEM supplemented with 15% FCS were harvested and Western blot analysis was performed. A: Ten micrograms of whole cell extracts was solubilized in SDS loading bu¡er. After SDS-PAGE, HA-tagged proteins were detected by immunoblotting with the 12CA5 monoclonal antibody. B: Fifty micro- grams of the same whole extracts was used for SDS-PAGE. Proteins were detected by immunoblotting with the a¤nity-puri¢ed polyclonal anti-MyoD antibody.

Article Snippet: After electrophoretic transfer of proteins onto nitrocellulose membranes, immunodetection was performed with the 12CA5 monoclonal antibody (dilution: 1/1000, Boehringer) or the MyoD antibody (dilution: 1/1000, Santa Cruz).

Techniques: Expressing, Transfection, Western Blot, SDS Page