Hes1 ab71559

Protein samples for western blot analysis were extracted by RIPA buffer (Thermo Scientific, Cat# 89900) plus a protease inhibitor cocktail (Roche, Cat# 11836153001) and phosphatase inhibitors (50 mM NaF, 2 mM Na₃VO₄, 4 mM sodium pyrophosphate). Commercially available antibodies were from the following sources: MYPT1 (2634 S), phospho-T853 MYPT1 (4563 S) and PDK1 (3062) from Cell Signaling Technology (MA, USA); MLC2 (PA5-17624) and phospho-Ser20 MLC (PA5-17727) from Thermo Scientific Pierce (IL, USA); ROCK1 (sc-5560) and Rnd3 (sc-53874) from Santa Cruz Biotechnology (CA, USA). Hes1 (ab71559) and Notch1 (ab27526) were from Abcam (MA, USA). Equivalent protein loading was verified by the intensity of a GAPDH (sc20357, Santa Cruz, CA) blot.

Cisplatin (P-4394), α-tubulin (T5168), anti-Notch3 (HPA044392—that exclusively detects the Valine-1744 residue of NICD3 (notch intracellular domain3) fragment), anti-mouse (12–349), and anti-rabbit (A6154) HRP tagged, Verapamil (V4629) and Gamma-secretase inhibitor ((GSI: N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT)) (D5942) were purchased from Sigma-Aldrich (St. Louis, MW, USA). IHC detection kit (ab236466), Jagged1 (ab77751) and Hes1 (ab71559) antibodies were from Abcam (Cambridge, UK). Pbx1/2 (sc-28313) and p21 (2947S) antibodies were from Santa-Cruz (sc-28313) and Cell Signaling Technology. The 17-mer rh-Jag1 peptide (CDDYYYGFGCNKFCRPR) (188–204), corresponding to amino acids 187–203 of human Jagged1 peptide, was purchased from Ana spec (Fermont, CA, USA). Dye cycle violet (DCV) was purchased from Invitrogen (Waltham, MA, USA).