Pan 14 3 3 antibody

Cell pellets were lysed in lysis buffer (20mM Tris (pH8), 150mM NaCl, 0.5% NP-40, 1mM EDTA) supplemented with protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail; Roche) and phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044; Sigma). Lysates were heated at 70°C for 10min in NuPAGE LDS sample buffer. Proteins were separated by SDS-PAGE on a Bolt 12% Bis-Tris Plus or a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane in NuPAGE transfer buffer plus 7.5% methanol. The membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBST) plus 5% milk powder then incubated with primary antibodies: pan-14-3–3 antibody (8312S, Cell Signaling Technology), V5 antibody (R96025, Invitrogen), 14-3-3γ (Cell Signaling Technology, 5522), FOXO3a (Cell Signaling Technology 12,829), FLAG (Sigma, F1804), HSP90 (Cell Signaling Technology, 4877S), pan-AKT (Cell Signaling Technology, 2920S), AKT-pSer473 (Cell Signaling Technology, 4060S), FOXO3a-pSer253 (Cell Signaling Technology, 9466S), beta-actin (Sigma Aldrich, A5441), YAP (Cell Signaling Technology, 12395S), p53 (Santa Cruz, sc-126). Membranes were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies and developed using Amersham ECL Western Blotting Detection Reagents.

Cell culture, S1P stimulation, and fluorescence imaging of the fixed and live cells were previously described with minor modifications [30 (link)]. The phosphorylation of Y⁴⁶⁴ in nmMLCK was detected by rabbit polyclonal antibody to pY⁴⁶⁴ in nmMLCK1 (Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 546 (Life Technologies, Grand Island, NY). Actin was detected by Alexa Fluor 633 Phalloidin (Invitrogen, Carlsbad, CA). Pull-down of nmMLCK was performed using anti-Flag M5 antibody (Sigma-Aldrich, MO) or anti-GFP antibody (Invitrogen, NY) as appropriate. 14-3-3 proteins were detected using pan-14-3-3 antibody (Cell Signaling Technology, MA).