Anti β actin

Primary splenic B cells or cultured cells were lysed by 2× Lysis buffer. Proteins were extracted by 10% Bis-Tris PAGE (Life technologies) with the cocktail of proteinase inhibitors (Life technologies) in it, transferred to polyvinylidene fluoride (PVDF). The electroblotted membranes were blocked by TBST containing 5% non-fat milk (BD) and were probed with anti-PTEN, anti-β-actin, anti-p-AKT 473, or anti-p-FoxO1/3a primary antibodies overnight at 4°C then immunoblotted with HRP-conjugated secondary antibodies (Dako). PTEN (138G6) Rabbit antibody (#9559), Phospho-AKT (Ser473) rabbit antibody (#4060), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) rabbit antibody (#9464), and β-actin (13E5) rabbit antibody (#4970) were purchased from Cell Signaling Technology. AID rabbit antibody was a kind gift from Dr. Feilong Meng (Shanghai Institute of Biochemistry and Cell Biology, China).

Cell and muscle samples corresponding to each experimental condition were trypsinized from the culture dish and homogenized with a lysis buffer containing 40 mM glycophosphate, 40 mM NaF, 10 mM EDTA and 20 mM of β-mercaptoethanol (final pH=6). The samples were placed in boiling water for 3 min and centrifuged at 9500 g for 3 min, before 100 μg of protein was applied to each lane. Unspecific binding sites on the blots were blocked by incubation in 5% low-fat dried milk powder in a phosphate buffered saline. Thereafter, primary rabbit polyclonal antibody anti-PYGB (kindly provided by Dr K. Nowak, Harry Perkins Institute of Medical Research, Nedlands, WA, Australia), the primary goat anti-PYGM [kindly provided by Dr Martinuzzi, Istituto di Ricovero e Cura a Carattere Scientifico Eugenio Medea – Associazione ‘La Nostra Famiglia', Conegliano (Treviso), Italy], mouse anti-GAPDH (Ambion, Life Technologies) or anti-β-actin (Sigma-Aldrich) were added. Peroxidase-conjugated anti-rabbit (Jackson InmunoResearch, West Grove, PA, USA), peroxidase-conjugated anti-goat (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and peroxidase-conjugated anti-mouse (Dako, Glostrup, Denmark) secondary antibodies were applied when using anti-PYGB antibody, anti-PYGM antibody and anti-GAPDH and anti-β-actin, respectively.

Immunoblotting analyses were performed as previously described [27 (link)]. The primary antibodies used were: anti-ACSVL3 (1:1000) [20 (link)]; anti-β-actin (1:6000); anti-GFAP (1:500, DAKO, Carpinteria, CA, USA) and anti-Tuj1 (1:1000, EMD).
For immunofluorescence staining, neurosphere cells were collected by cytospin onto glass slides, fixed with 4% paraformaldehyde for 30 min at 4°C, permeabilized with PBS containing 0.5% Triton X-100 for 5 min and stained with anti-GFAP and anti-Tuj1 antibodies according to the manufacturers’ protocols. Secondary antibodies were conjugated with Alexa 488 or Cy3 (Life Technologies). Coverslips were placed with Vectashield antifade solution containing 4′6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Immunofluorescent images were analyzed using Axiovision software (Carl Zeiss, Microscope, Thornwood, NY, USA).