A5060

Cell extracts were prepared in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–12% gradient gels, Invitrogen) and subsequently transferred onto nitrocellulose membranes. Primary antibodies for MLH1 (554073, BD Pharmigen), MSH2 (ab52266, Abcam), MSH3 (ab69619, Abcam), Tubulin (3873, Cell Signalling) and ß-Actin (A5060, Sigma) were used at 1:1000. Secondary antibodies were used at 1:5000 (HRP-conjugated goat anti-mouse or anti-rabbit IgG from Jackson Immunochemicals). Immunoblots were imaged using a Curix 60 (AGFA) table-top processor. Uncropped immunoblots can be found in the Source Data file (for Fig. 3b) and Supplementary Fig. 5.

Electrophoretic analysis and immunoblots were performed following [20 (link)]. Lysates of control and treated cells were submitted to 13% SDS-PAGE and then protein transfer onto Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA) in a Novablot semidry apparatus (Pharmacia, Stockholm, Sweden). The primary antibodies used for immuno-detection were: rabbit polyclonal anti-Beclin-1 antibody (H-300, sc-11427, Santa Cruz Biotechnology, Dallas, TX, USA; working dilution 1:1000), rabbit polyclonal anti-LC3 antibody (L8918, Sigma; working dilution 1:750), rabbit polyclonal anti-p62/SQSTM1 (P0068, Sigma; working dilution 1:1000), and rabbit polyclonal anti-actin antibody (A5060, Sigma; working dilution 1:500). The latter antibody was used for a loading control. The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG antibody (NA934V, GE Healthcare; working dilution 1:2500). Protein bands were visualized in a Versadoc MP4000 molecular digital imaging system (Bio-Rad, Hercules, CA, USA) using the Immun-Star WesternC Chemiluminescent Kit (Bio-Rad), and analyzed by ImageJ software. Quantitation was performed after normalization of the signals with respect to those obtained with anti-actin antibody reaction.

Cerebral microvessels at 6 or 12 weeks were processed and analyzed with Western blot as described previously by us [28 (link), 56 (link)]. Briefly, the isolated brain microvessels as described above were homogenized and processed for Western blotting. Primary antibodies, including anti-TNF-α (1:1000, ab6671, Abcam, Cambridge, MA, USA), anti-IL-6 (1:1000, ab9324, Abcam, Cambridge, MA, USA), anti-MCP-1 (1:2000, ab25124, Abcam, Cambridge, MA, USA), anti-INF-γ (1:1000, ab7740, Abcam, Cambridge, MA, USA), anti-iNOS (1:2000, ab15323, Abcam, Cambridge, MA, USA), anti-VCAM-1 (1:5000, ab134047, Abcam, Cambridge, MA, USA), and anti-β-actin (1:5000, A5060, Sigma-Aldrich, St. Louis, MO, USA), were incubated on the membrane at 4°C overnight. Protein expression was detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).