Rabbit anti beclin 1 antibody

Minimum essential medium (α-MEM) and Dulbecco modified minimal essential medium (D-MEM) was obtained from Gibco Laboratories (Buenos Aires, Argentina). Fetal bovine serum (FBS) was purchased from Natocor S.A. (Córdoda, Argentina). The inhibitors α-difluoromethylornithine (DFMO) and Chloroquine (CQ) and the TRITC-conjugated phalloidin was purchased from Sigma (Buenos Aires, Argentina). The rabbit anti-Beclin-1 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, INC) and the polyclonal rabbit anti-LC3 antibody from Sigma (Buenos Aires, Argentina). The following antibodies were also used: anti-Atg5 (Abcam), anti-p62 (Abcam), Anti-ubiquitin (Santa Cruz), anti-β-actin (Genescript) and the anti-β tubulin (E7) was obtained from Developmental Studies Hybridoma Bank. The secondary antibodies Cy3-conjugated anti-mouse and Cy3-conjugated anti-rabbit were purchased from Jackson Immuno Research Laboratories, INC, as well as the antibodies HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG and HRP-conjugated Anti-goat IgG. Hybond-ECL nitrocellulose membranes were from Amersham. The DNA marker Hoechst 33342 was purchased from Life Technologies.

Western blotting analysis was performed as described previously [22 (link)]. Antibodies used in this experiment were as follows: goat anti-COTE1 (1 : 200; Santa Cruz Biotechnology, CA, USA), rabbit anti-LC3A/B antibody (1 : 1000; Cell Signaling Technology, Massachusetts, USA), mouse anti-P62 antibody (1 : 1000; Cell Signaling Technology, Massachusetts, USA), rabbit anti-Beclin-1 antibody (1 : 200; Santa Cruz Biotechnology, CA, USA), and anti-β-actin (1 : 500; Santa Cruz Biotechnology, CA, USA). The gray value of proteins was quantitatively analyzed by ImageJ (v.1.8.0).

Immunoprecipitation of endogenous Beclin 1 was performed using a polyclonal goat anti-Beclin 1 antibody (Santa Cruz Biotechnology; 1:50 dilution) and immunoprecipitation of Flag-tagged Beclin 1 was performed using a monoclonal anti-Flag M2 antibody pre-conjugated to agarose (Sigma–Aldrich; 1:20 dilution). For co-immunoprecipitation of Beclin 1 and BCL2, immunoprecipitation was performed using a polyclonal rabbit anti-Beclin 1 antibody (Santa Cruz Biotechnology; 1:50 dilution) and BCL2 was probed with a monoclonal HRP antibody (Santa Cruz Biotechnology; 1:200 dilution).