Starbright 700

For Western blotting, the following antibodies were used: rabbit anti-cystatin F (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-N-terminal part of cystatin F (Amsbio, Abingdon, UK), rabbit anti-β-actin (Sigma-Aldrich), rabbit and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, Rosemont, IL, USA), rabbit anti-C/EBPα, mouse anti-cathepsin C, mouse anti-granzyme A, mouse anti-granzyme B, mouse anti-perforin, and mouse anti-legumain (B-8) (all from Santa Cruz Biotechnology, Dallas, TX, USA). The mouse anti-cathepsin H antibody 1D10 [87 (link)] and sheep anti-cathepsin L antibody [88 (link)] were prepared as described previously. The following secondary antibodies were used: anti-rabbit, anti-mouse, and anti-sheep secondary antibodies conjugated with horseradish peroxidase (HRP) (Jackson Immuno Research, West Grove, PA, USA); anti-rabbit and anti-mouse secondary antibodies conjugated with the fluorescent dyes DyLight 650 and DyLight 550, respectively (Invitrogen, Carlsbad, CA, USA); and anti-mouse secondary antibody conjugated with fluorescent dye StarBright 700 (Bio-Rad, Hercules, CA, USA).

To determine the steady-state level of the hPanKs isoforms, all the mutant strains Δcab1 expressing PANK1, PANK2 or PANK3 were pregrown in SC glucose 2% medium and then inoculated in SC medium supplemented with 2% galactose until 2–3 OD₆₀₀ was reached at 28 °C or 37 °C depending on the mutated strain analyzed. Then, 10 OD₆₀₀ cells for each strain were collected and total protein extraction was performed using the TCA method by chilling the cells in the presence of 20 mM NaOH, 0.5% β-mercaptoethanol, 650 µM PMSF and 25% TCA on ice. The protein extracts were then resuspended in 150 μl of Laemmli Sample Buffer at a pH of 6.8. An equal amount of protein suspension, corresponding to 1 OD₆₀₀ of the original cell suspension was loaded on 12% SDS-PAGE and electroblotted on a nitrocellulose filter. The filter was incubated with mouse α-Myc (1:2000) and mouse α-Por1 (1:10,000) primary antibodies. Secondary antibody anti-mouse StarBright 700 (BioRad, Hercules, CA, USA, 1:10,000 dilution) was used to detect both primary antibodies. Fluorescent signals were revealed using Bio-Rad ChemiDoc Imagers and analyzed with Image Lab Software (Bio-Rad). The ratios between Myc and Por1 were calculated.

FLAG-tagged Tg in pcDNA3.1+/C-(K)-DYK plasmid was purchased from Genscript (Clone ID OHu20241). Site-directed mutagenesis was then performed to generate FT-G2341R, FT-L2284P, FT-C1264R, FT-A2234D, and untagged Tg plasmids (supplemental Table S1). Primary antibodies were acquired from commercial sources and used at the indicated dilutions in immunoblotting buffer (5% bovine serum albumin in Tris-buffered saline pH 7.5, 0.1% Tween-20, and 0.1% sodium azide). Mouse monoclonal antibodies were used for the detection of KDEL (1:1000, Enzo Life Sciences, ADI-SPA-827), M2 anti-FLAG (1:1000, Sigma Aldrich, F1804). Polyclonal rabbit antibodies were used to detect Calnexin (1:1000, GeneTex, GTX109669), protein disulfide isomerase family A member 4 (PDIA4) (1:1000, Proteintech, 14712-1-AP), DNAJC10 (1:500, Proteintech, 13101-1-AP), thyroglobulin (1:1000, Proteintech, 21714-1-AP), UGGT1 (1:1000, Proteintech, 14170-1-AP), STT3A (1:2000, Proteintech, 12034-1-AP), and STT3B (1:2000, Proteintech, 15323-1-AP). Secondary antibodies were obtained from commercial sources and used at the indicated dilutions in 5% milk in Tris-buffered saline pH 7.5, 0.1% Tween-20 (TBS-T): Goat anti-mouse Starbright700 (1:10,000, Bio-Rad,12004158), Goat anti-rabbit IRDye800 (1:10,000, LI-COR, 926-32211), and Goat anti-rabbit Starbright520 (1:10,000, Bio-Rad,12005869).