Ab133757

Brain-tissue samples were collected using 2-deoxy-d-glucose, ethylene glycol tetra-acetic acid (EGTA), and reduced glutathione. Western blot and immunofluorescence analyses involved primary antibodies; primary monoclonal rat anti-tyrosine hydroxylase (T1299, Sigma-Aldrich, St. Louis, MI, USA), monoclonal rabbit anti-NPY5R (AB133757, Abcam, Cambridge, UK), polyclonal rabbit AMPKα1/2 (H-300) (sc-25792, Santa Cruz, CA, USA), polyclonal rabbit anti-phospho AMPKα (THR172, Millipore, Burlington, MA, USA), monoclonal mouse anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA), and polyclonal rabbit anti-tyrosine hydroxylase phosphoSer40 (AB5935, Millipore, Burlington, MA, USA). Primary antibodies in Western blot analysis were detected using polyclonal goat anti-mouse IgG HRP (HAF007, R&D Systems, Minneapolis, MN, USA) and polyclonal goat anti-rabbit IgG (H + L) HRP (65-6120, Invitrogen, Waltham, MA, USA), whereas Alexa Fluor 488 conjugated with goat anti-rabbit IgG (H + L) secondary antibody (A11008, Thermo Fisher Scientific, Waltham, MA, USA) and CY3 conjugated with goat anti-mouse IgG (H + L) secondary antibody (A10521, Thermo Fisher Scientific, Waltham, MA, USA) were used in immunofluorescence.

Proteins were extracted from cell and xenograft tumors and processed for SDS-polyacrylamide gels electrophoresis and Western blot analysis as described previously [12 (link)]. Primary antibodies used were anti-human integrin α1/CD49A (1:2000, AF5676, R&D System, Minneapolis, MN, USA), anti-human integrin α2 (1:10,000, Ab 133757, Abcam, Toronto, ON, Canada) and anti-β-actin (1:80,000, C4, Millipore, Etobicoke, ON, Canada). Secondary antibodies were horseradish peroxidase-conjugated anti-sheep (12-342, Millipore), anti-rabbit (NA934V, Amersham, Mississauga, ON, Canada) and anti-mouse (NA913V, Amersham) antibodies. All experiments were repeated a minimum of three times.

Cells were cultured on glass cover slips, fixed in 4% (w/v) paraformaldehyde and stained with rabbit polyclonal anti-Y5R (ab133757, Abcam; 1:250) and mouse monoclonal anti-RhoA-GTP (NewEast Biosciences King of Prussia, PA; 1:100) antibodies. DNA was stained using DAPI at 0.5 µg/ml in PBS.