Anti mouse irdye 800

S. cerevisiae strains used in this study are described in Table S1. Anti-HA monoclonal antibody (HA.11, raised in mouse) and anti-myc monoclonal antibody (9E10 c-myc, raised in mouse) were purchased from Covance. Anti-Kar2 and anti-Sec61 antibodies (rabbit) were provided by P. Walter (University of California, San Francisco, San Francisco, CA). Anti-Sis1 antiserum (rabbit) was a gift from D. Cyr (University of North Carolina, Chapel Hill, NC). Monoclonal antiproteasome 20S α (mouse) and polyclonal anti–histone H3 (rabbit) were purchased from Abcam. Monoclonal anti–3-phosphoglycerate kinase antibody (mouse) and monoclonal anti-V5 antibody (mouse) were purchased from Invitrogen, monoclonal anti-Ydj1 antibody (mouse) was from StressMarq, monoclonal anti-GFP antibody (mouse) was purchased from Roche, and monoclonal anti-FLAG (mouse) was purchased from Sigma-Aldrich. Secondary antibodies labeled with Alexa Fluor 488 (anti–mouse) or Alexa Fluor 596 (anti–rabbit) were purchased from Molecular Probes. Anti–rabbit IRDye 680 and anti–mouse IRDye 800 secondary antibodies were purchased from LI-COR Biosciences. Anti-Ubr1 antiserum was raised in rabbit against a recombinant protein containing the 100 N-terminal amino acids of Ubr1. Anti-San1 antiserum was raised in rabbit against a protein containing the 100 C-terminal amino acids of San1.

Protein G functionalized Dynabeads (Thermo Fisher Scientific) were used to probe binding of PKA-II holoenzymes to the phospho-RII antibodies. Anti–phospho-RII (S99) antibodies (∼2 µg input; Abcam) were captured following the manufacturer’s instructions, washed twice in HBS-P buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% P20 surfactant), and incubated for 20 min with purified, preformed PKA-II holoenzyme (Cα:RIIβ, 3.6 µM:3 µM) in HBS-P buffer. The buffer was supplemented with 1 mM ATP, 10 mM MgCl₂, and 0.5 or 10 µM cAMP. The beads were washed four times and transferred into new tubes. Elution was performed using 10 mM glycine, pH 2.2, and SDS sample buffer. The samples were subjected to SDS-PAGE and proteins were detected by Western blotting using Cα (sc-28315; Santa Cruz) and RIIβ (610625; BD Transduction Laboratories) on separate Western blots. Detection and signal quantification was performed using the Odyssey Fc Imager (Li-Cor) with the fluorescently labeled anti–mouse IRDye800 (Li-Cor) secondary antibody or mouse IgG κ-binding protein CFL790 (sc-516181; Santa Cruz).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, with 1 mM phenylmethylsulfonyl fluoride in isopropanol added right before lysis) with constant agitation at 4°C for 30 min. Lysates were then centrifuged at 12,000 rpm at 4°C for 20 min, and total protein extracts were boiled in Laemmli sample buffer (BioRad), separated by SDS–PAGE under reducing conditions, and transferred to polyvinylidene difluoride membranes (Millipore), which were then incubated with the following primary antibodies: anti-GAPDH (1:2000; GeneTex GT239), anti-Nav1 (1:1000), or anti-TrkB (1:500; BD Biosciences 610101). Immunoreactive bands were visualized by the LI-COR Odyssey Imaging System using anti-mouse IRDye 800 and anti-rabbit IRDye 680 (1:5000; LI-COR). To quantify and compare signal intensities, a box was applied to encompass the band of interest, and integrated intensity was determined after background subtraction. In the case of TrkB, we assumed that both bands of the doublet appearing in the WT and SH-SY5Y cell extracts corresponded to TrkB and were included in the boxed region.