Criterion tgx protein gel

Cells were pre-cultured overnight, diluted to OD₆₀₀ = 0.1, and grown until mid-exponential phase (OD₆₀₀ ~ 0.5) in YPD at 22 °C (Hsp70 mutant strains and control) or 30 °C. Cells were harvested as 2 OD₆₀₀ units, resuspended in 200 µl of 0.2 M NaOH, and incubated for 20 min on ice. Cell pellets were resuspended in 50 µl Laemmli sample buffer (Bio-Rad) supplemented with β-mercaptoethanol and boiled for 5 min at 95 °C. 25 µl supernatant was typically loaded per lane on a 4–15% precast polyacrylamide 18 well Criterion TGX protein gel (Bio-Rad). Gel transfer onto PVDF membranes was performed using a wet blotting system (Criterion, Bio-Rad). Membranes were probed with anti-GFP (ab6556, Abcam, 1/10,000 dilution) and anti-Pgk1 (ab90787, Abcam, 1/10,000 dilution) as a loading control overnight at 4 °C. As secondary antibodies, goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD were used (LI-COR; 1/20,000 dilution) and membranes were scanned using the LI-COR Odyssey Infrared scanner. Hsp104-GFP-Pea2 protein levels were calculated by measuring the background corrected band intensities using Fiji/ImageJ 2.1.0/1.53c software and normalizing to Pgk1 protein levels. The adjusted Hsp104-GFP-Pea2 protein level of the respective control strain was set to 1. Four independent experiments were performed. Uncropped Western blots are shown in Supplementary Fig. 8.

Protein lysates were collected 48 hours after transient transfection of EMG or cDNA plasmids into HEK293 cells. 40μg of lysate were loaded per sample into a 7.5% Criterion TGX protein gel (BioRad). Transfer to PVDF membrane was performed in a Trans-Blot Turbo Transfer System (BioRad). After blocking, the membrane was probed with either mouse monoclonal anti-CFTR antibody 596 binding amino acids 1204–1211 (CFFT, University of North Carolina Chapel Hill) or mouse monoclonal anti-CFTR antibody 570 binding amino acids 731–742 (CFFT, University of North Carolina Chapel Hill) diluted to 1:5,000. Rabbit monoclonal anti-sodium/potassium-ATPase (Abcam) diluted 1:50,000 was used as a loading control. Secondary antibodies were anti-mouse (1:150,000 GE Healthcare) and anti-rabbit (1:100,000 GE Healthcare), respectively. Blots were exposed on film using ECL Primer Western Blotting Detection Reagent (GE Healthcare).

Twenty-five ml volumes of culture for each transformant to be analyzed were grown in Friis broth. Cells were pelleted by centrifugation (10,000 × g for 20 min, 4°C) and washed twice with phosphate buffered saline (PBS). The final cell pellet was resuspended in water at a 1:100 volume and protein determined by Qubit and adjusted to 1 mg/ml with water. An equal volume of 2X Laemmli Sample Buffer (BioRad, Hercules, CA, United States) was added, the samples were boiled for 5 min and centrifuged in a microfuge for 3 min prior to loading on 8–16% Criterion TGX Protein gels (BioRad). Proteins were transferred to nitrocellulose using a BioRad Criterion^TM blotter. Blots were blocked with 2% fish gelatin (Sigma Chemical Co.) in TS buffer (10 mM Tris–150 mM NaCl, pH 7.2) for 2 h at room temperature and then reacted with a 1:500 dilution of monoclonal F1B6 (Zhang et al., 1995 (link)) in TST buffer (TS – 0.01% Tween 20) overnight at 4°C. The blots were washed with TST and then reacted with a 1:500 dilution of alkaline phosphate conjugated goat anti-mouse IgG, IgM (H + L; Invitrogen) in TST for 2 h, washed three times and developed with SIGMAFAST Fast Red TR/Naphthol substrate (Sigma Chemical Co.).