Anti tubulin clone b5 1 2

Membranes were decorated using anti‐mCherry‐1C51 (Abcam), anti‐HA.11‐16B12 (BioLegend), anti‐G6PDH (Sigma‐Aldrich), anti‐Tubulin (clone B5‐1‐2, Sigma‐Aldrich), anti‐WIPI1 (W2394 Sigma‐Aldrich), anti‐Tubulin (T9026 Sigma‐Aldrich) anti‐Vps35 (ab10099 Abcam, ab157220 Abcam) and anti‐Vps26 (ab181352 Abcam). After incubation with the primary antibody, signals were detected by secondary antibodies coupled to infrared dyes (LICOR), IRDye® 800CW goat anti‐mouse IgG, IRDye® 800CW goat anti‐rabbit IgG, IRDye® 680LT goat anti‐rabbit IgG, IRDye 800CW goat anti‐rat IgG, IRDye® 680RD donkey anti‐rabbit IgG, and detected on a LICOR Odyssey Infrared Imager. Band intensity was quantified using Odyssey software with background removal activated.

Lysates and eluates from Immunoprecipitations were run on 10% acrylamide gels for SDS–PAGE, freshly prepared and used the same day: 10% Protogel (30% w/v acrylamide, 0.8% bis‐acrylamide (37.5:1 solution, National diagnostics, Atlanta, USA), 380 mM Tris–HCl pH 8.8, 0.1% w/v SDS (Applichem, Darmstadt, Germany), 0.06% v/v TEMED (Applichem), 0.06% w/v APS (Applichem) for the running gel and 5% Protogel, 165 mM Tris–HCl pH 6.8, 0.1% w/v SDS, 0.08% v/v TEMED, 0.04% w/v APS for the stacking gel. Running buffer for SDS–PAGE was 190 mM glycine (Applichem), 25 mM Tris‐base (Applichem), 0.5% SDS. To facilitate Atg18 migration and avoid formation of aggregates, samples were reduced and denatured at 90°C using NuPAGE buffer (Thermo Fisher) containing LDS instead of SDS and supplemented with 100 mM DTT. Gels were blotted on 0.45 µm nitrocellulose membrane (Amersham) overnight at a constant current of 200 mA using a Trans‐Blot® Cell (Bio‐Rad, USA). Membranes were decorated using anti‐mCherry‐1C51 (Abcam), anti‐HA.11‐16B12 (BioLegend), anti‐G6PDH (Sigma‐Aldrich), anti‐Tubulin (clone B5‐1‐2, Sigma‐Aldrich), and anti‐WIPI1 (C‐terminal epitope, Sigma‐Aldrich).

We used affinity-purified, subunit-specific polyclonal antibodies (Abs), produced in rabbit against peptides derived from the C-terminal (COOH), N-terminal (NH) of mouse and AMPAR GluA1 and GluA2/3 subunits. The Ab against the GluA2/3 subunit was directed against the C-terminus peptide (EGYNVYGIESVKI). The Ab against the GluA1 subunit was directed against the extracellular domain peptides (RTSDSRDHTRVDWKR) corresponding to aminoacids 253–267 (271–285 if numbered from the signal peptide), this region is not conserved in GluA2-4, nor Kainate and NMDAR. GluA1 and GluA2/3 sequences were the same as those reported by Chemicon International. The specificity of the affinity-purified Abs was previously tested by western blotting studies using cells transfected and non-transfected with GluA1 and GluA2/3. Our tests do not show crossreactivity between GluA1 and GluA2/3 Abs, as it has been reported in the specificity tests of Chemicon International.
Anti GluN1 (clone 54.1) was from BD Pharmigen, anti GluN2A (clone A3-2D10) was from Invitrogen, anti-GluN2B (clone N59/20) was from Antibodies Incorporated, anti-tubulin (clone B-5-1-2) was from Sigma-Aldrich and anti Na/K ATPase was described in [26 (link)].