Irdye 680rd goat anti rabbit igg antibody

Cells were washed twice with cold PBS and lysed with RIPA buffer containing protease and phosphatase inhibitors (ThermoFisher Scientific). The polyvinylidene difluoride membranes were incubated with specific antibodies, including CREBRF rabbit monoclonal antibody (mAb) (ThermoFisher, PA5-68552; 1:1000), CREB3 rabbit mAb (Proteintech, 11275-1-AP; 1:1000), NR3C2 mouse mAb (Novus, NB300-562; 1:1000), c-Myc rabbit mAb (Novus, NB600-336; 1:1000), ATG5 rabbit mAb (Novus, NB110-53818SS; 1:1000), β-catenin mouse mAb (Novus, NBP1-54467SS; 1:1000), and β-actin mouse mAb (Sigma-Aldrich, A3854; 1:1000). The membranes were incubated with IRDye^® 680RD Goat anti-Rabbit IgG Antibody (LI-COR Biosciences, 926-68071; 1:5000) or IRDye^® 800CW Goat anti-Mouse IgG Antibody (LI-COR Biosciences, 926-32210; 1:5000). Protein bands were measured using an Odyssey Infrared Imaging System (ODYSSEY CLx, Li-COR). The fluorescence intensity was evaluated using Image Studio Lite version 5.2.

PIP strips (Echelon Biosciences) were used to assess binding of purified FYVE domains to indicated lipids. Membrane was incubated for 1 h at room temperature in Tris-buffer saline with 0.1% Tween 20 (TBST) plus 3% fatty-acid (FA)-free BSA (Akron Biotechnology). The indicated concentration of protein in TBST plus 3% FA-free BSA was incubated at room temperature for 1 h. Membrane was washed three times in TBST plus 3% FA-free BSA for 10 min each between protein and antibody incubations. The primary antibody was incubated at 4°C overnight: rabbit anti-His6 (1:1000, ab137839, Abcam). The secondary antibody was incubated for 1 h at room temperature: IRDye 680RD goat anti-rabbit-IgG antibody (926-68171, Li-Cor, Lincoln). Membranes were imaged using the ChemiDoc MP Imaging System (Bio-Rad).

Protein extracts were prepared as previously described (Millen et al., 2009 (link)). Briefly, cells were lysed on ice by resuspension in 1 ml cold H₂O supplemented with 150 µl 1.85 M NaOH and 7.5% (v/v) β-mercaptoethanol. After a 10-min incubation on ice, the protein was precipitated by addition of 150 µl 50% (w/v) trichloroacetic acid. Pellets were washed twice with acetone, resuspended in 100 µl 1× SDS-PAGE buffer, and boiled for 5 min at 95°C. Primary antibodies were incubated overnight at 4°C and were as follows: anti-GFP (1:1000, ab290, Abcam), anti-PGK1 (1:1000, ab113687, Abcam), anti-Rps6 (1:1000, ab40820, Abcam), anti-phospho-Rps6 (1:1000, 4858, Cell Signaling Technology, Danvers). Secondary antibodies were incubated for 1 h at room temperature and were as follows: IRDye 680RD goat anti-rabbit-IgG antibody (926-68171, Li-Cor, Lincoln) and IRDye 680RD goat anti-mouse-IgG antibody (926-68070, Li-Cor). These were detected using the ChemiDoc MP Imaging System (Bio-Rad). Bands were integrated and quantified using Fiji. See Fig. S7 for original blots.