Anti ha clone c29f4

Cells were lysed in pH 7.5 buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet-P40 and 2 mM Na4P2O7 plus protease inhibitors (Roche). Cell lysates were loaded on NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (NP0321BOX) and running buffer (Life Technologies) using standard protocols. Blotting was done on a PVDF membrane (Invitrolon, LC2005 Thermo Fisher) using transfer buffer (Life Technologies). The following antibodies were used: anti-LAMP2 (clone H4B4, Santa Cruz Biotechnology), anti-HA (clone C29F4, Cell Signaling), anti-CTSC (clone D-6, Santa Cruz Biotechnology), anti-mTOR (2972, polyclonal, Cell Signaling), anti-pS6 Ser235/236 (polyclonal, Cell Signaling, 2211S), anti- GAPDH (clone 14C10, HRP conjugate, Cell Signaling, 3683S) and anti-β-actin (8H10D10, HRP conjugate, Cell Signaling). Signals were detected using HRP conjugated secondary antibodies (Cell Signaling) followed by enhanced chemiluminescence (ECL, GE Healthcare Life Science) and were recorded on a Biorad Chemidoc imaging system.

Western blotting was performed as previously described39 (link). The conditions and duration of treatments of the cells before protein isolation are described in the text and the figure legends. Primary antibodies included p-ERK Thr204/205, ERK, p-MEK Ser217/221, MEK, anti-HA (clone C29F4) and GAPDH (all from Cell Signaling Technology, Danvers, MA), BRAF (H-145) and CRAF (E-10) (both from Santa Cruz biotechnology, Santa Cruz, CA), anti-MYC and anti-actin (Sigma Aldrich, St. Louis, MO). Secondary antibodies were HRP conjugates (Santa Cruz biotechnology, Santa Cruz, CA). Densitometry of bands was performed by using ImageJ program.

Free-floating sections of the medulla oblongata were processed using Duolink (Sigma-Aldrich, St. Louise, MO, USA) according to the manufacturer’s protocols as described previously [24 (link),25 (link),26 (link)]. HA and α-syn PLA probes were generated by linking the plus and minus oligonucleotides with primary antibodies anti-HA (clone C29F4, Cell Signaling) and anti-human α-syn (clone syn211, Millipore, Burlington, MA, USA) using a probemaker kit (Duolink, Sigma-Aldrich, Janvier Labs). HA/HA and α-syn/α-syn interactions were detected using a direct PLA method, which required incubating sections overnight in solutions containing the plus and minus PLA probes directly conjugated to the HA (1:250) or human α-syn–antibody (1:100; mouse-anti-Syn211, Millipore, Burlington, MA, USA). Samples treated only with the plus probe served as negative controls. Following ligation and amplification, specific PLA signals were visualized using a brightfield detection kit (Duolink, Sigma-Aldrich, St. Louise, MO, USA). Sections were mounted on object slides and coverslipped using histomount mounting media (Life Technologies, Carlsbad, CA, USA).