Ab37500

Total protein was extracted using RIPA buffer (KeyGen, China). Protein concentration was quantified using a BCA protein kit (Beyotime, China). Proteins (30 µg) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% milk, membranes were incubated with primary antibodies overnight at 4°C, including Numb (ab155415, Abcam, USA), Numbl (ab37500, Abcam, USA), Notch1 (ab128076, Abcam, USA), Hes1 (ab71559, Abcam, USA), and β-actin (ab8227, Abcam, USA). Subsequently, the samples were incubated with secondary antibodies (Abcam, USA) for 1.5 h at room temperature and analyzed using enhanced chemiluminescence reagent (Beyotime, China) [28 (link)].

MM cells were collected and washed twice with -cold PBS, and incubated for 10 min at 4 °C in Triton X-100 lysis buffer (30 mM Tris-HCl pH 7.5, 150 mM NaCl, 25 mM NaF, 1% Triton X-100, 10% glycerol, 2 mM Naorthovanadate). MM Cells were immobilized for 20 min with cold PBS containing 4% formaldehyde (Sigma Aldrich), permeabilized with 0.1% Triton X-100 for 10 min, and then incubated for 2 h at 4 °C in 1% BSA. Then the cells were incubated with primary antibodies at 4 °C overnight. After washing in PBS, cells was incubated with appropriate sheep FITC- or TRITC- conjugated secondary antibodies (1:250; Jackson ImmunoResearch Laboratories) for 2 h at RT. Sections were washed in PBS and counterstained with DNA stains using 4′6-diamidino-2-phenylindole dihydrochloride (Dapi, Sigma). Finally, cells were reversed on glass slides with glycerol and PBS (1:1). Immunolabeled cells were examined under a Leica Confocal Laser Scanning Microscope and fluorescence microscope. The following antibodies were used for immunofluorescence: anti-integrin-β1 at 1:300 (MAB1981, Chemicon); anti-Numbl at 1:200 (ab37500, abcam).

Protein extracts for Western blot analysis were obtained as described previously [17 (link)]. For Western blot detection, we used NUMB (ab4147, Abcam, 1 μg/mL) and NUMBL (ab37500, Abcam, 1 μg/mL) antibodies. α-Tubulin (T9026, Sigma) was used as a control. Horseradish peroxidase-labeled rabbit anti-mouse (ab97046, Abcam, diluted 1:5,000) and goat anti-rabbit (ab97051, Abcam, diluted 1:5,000) secondary antibodies were used.