Anti ulk1 antibody

Total protein lysates were extracted from animal tissues or cells using a Triton-based cell lysis buffer (40 mM HEPES, 120 mM NaCl, 1 mM EDTA, 1.5 mM Na₃VO₂, 50 mM NaF, 10 mM β-glycerophosphate, 20 mM MoO₄, 0.5% Triton X-100, protease inhibitor, phosphatase inhibitor). The lysates were incubated with anti-ULK1 antibody (Santa Cruz Biotechnology; sc10900), or anti-VCP antibody (Santa Cruz Biotechnology; sc-57492) overnight at 4°C and precipitated with protein G agarose beads (Thermo Fisher Scientific; 20399). DDK IP was performed using anti-FLAG M2 affinity gel according to the manufacturer’s instructions (Sigma Aldrich; A2220). Eluate was electrophoretically separated on 4%−12% Bis-Tris gels (Life Technologies; NP0335BOX). Proteins were then transferred to PVDF membranes followed by standard immunoblot analyses as described above.

Endogenous ULK1 was extracted from the hippocampi of 4-wk-old WT mice or from MEFs by using a Triton-based cell lysis buffer (40 mM HEPES, 120 mM NaCl, 1 mM EDTA, 1.5 mM Na³VO⁴, 50 mM NaF, 10 mM β-glycerophosphate, 20 mM MoO⁴, 0.5% Triton X-100, protease inhibitor, phosphatase inhibitor). The lysates were incubated with anti-ULK1 antibody (Santa Cruz Biotechnology, sc10900) overnight at 4°C and precipitated with Protein G agarose beads (Thermo Scientific). For immunoprecipitation of GFP–SEC16A, whole-cell extracts were prepared from 293T cells by using the described Triton-based buffer and precipitated with anti-GFP antibody–conjugated sepharose beads (Abcam, ab69314) after overnight incubation at 4°C. The beads were washed 5 times with cold Triton-based buffer and incubated at 95°C for 5 min in SDS sample buffer (Sigma Aldrich). Phosphatase treatment was performed on GFP IPs by using calf alkaline intestinal phosphatase (Sigma Aldrich) per the manufacturer’s protocol, before elution in SDS sample buffer. Anti–FLAG M2-agarose beads (Sigma Aldrich, A2220) were used for immunoprecipitation of FLAG-tagged proteins.