Rabbit anti flag

Cells were washed with Hanks buffered salt solution (HBSS, Sigma-Aldrich, St. Louis, MO, USA), then lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz, Dallas, TX, USA) supplemented with Halt protease inhibitor (Pierce, Rockford, IL, USA) and benzonase (Millipore Sigma, St. Louis, MO, USA) for 3–5 min at room temperature, and then spun at max speed (14,800 rpm) at 4 °C for 10 min. The soluble supernatant was collected and protein concentration was quantified via bicinchoninic assay (BCA) (Pierce). Twenty to thirty µg of soluble supernatant or 20 μL of conditioned media was run on a 4–20% Tris-Gly SDS-PAGE gel (Life Technologies) and transferred onto a nitrocellulose membrane using an iBlot2 device (Life Technologies). After probing for total transferred protein using Ponceau S (Sigma-Aldrich), membranes were blocked overnight in Odyssey Blocking Buffer (LICOR, Lincoln, NE, USA). Membranes were probed with rabbit anti-FLAG (1:5000; Thermo Fisher Scientific, Waltham, MA, USA, cat# PA1-984B) or mouse anti-β-actin (1:1000; Sigma-Aldrich, cat# A1978). All Western blot imaging was performed on an Odyssey CLx (LI-COR) and band quantification was performed using Image Studio software (LI-COR).

Samples in Laemmli sample buffer were boiled at 95°C for 10 min. The indicated amount of each mixture was electrophoresed on NuPAGE 4% to 12% Bis-Tris gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes using the iBlot 2 transfer device (Thermo Fisher Scientific). Following transfer, membranes were blocked in 3% skim milk at room temperature for 2 h, incubated with the indicated primary antibodies for 1 h, washed 3 times, incubated with secondary antibodies (GE Healthcare and Promega) for 1 h, and washed 3 times. Rabbit anti-FLAG (Thermo Fisher Scientific) and rabbit anti-DnaK (Thermo Fisher Scientific) antibodies were used at 1:5,000 dilution in TBST. Mouse anti-TF (Takara Bio) antibodies were used at 1:50,000 dilution. Rabbit anti-S1 ribosomal protein (Agrisera) antibodies were used at 1:1,000 dilution. Horseradish peroxidase-conjugated anti-rabbit (GE Healthcare) and anti-mouse (Promega) secondary antibodies were used at 1:5,000 dilution. Chemiluminescent signal was developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and captured with an ImageQuant LAS 4000 imager (Fujifilm).

After the addition of Laemmli Sample Buffer (Cat. No. 1610747, Bio‐Rad) including NuPAGE™ Sample Reducing Agent (10X) (Cat. No, NP0009, Thermo Fisher), cell lysates were separated on 4–20% Mini‐PROTEAN TGX Stain‐Free™ protein gels (Cat. No. 4568095, Bio‐Rad, Hercules, CA, USA), and transferred to Trans‐Blot Turbo Mini 0.2 μm Nitrocellulose membranes (Cat. No. 1704158, Bio‐Rad). Membranes were blocked in 5 mL EveryBlot Blocking Buffer (c#. 12010020, Bio‐Rad) for 30 min. Incubation with primary antibodies diluted in blocking buffer, including rabbit anti‐APR3 (Cat. No. PA5‐62388, ThermoFisher, 1/1000 dilution), rabbit anti‐Flag (Cat. No. PA1‐984B, ThermoFisher, 1/1000 dilution), and rabbit anti‐GAPDH (Cat. No. G9545, Sigma‐Aldrich, 1/5000 dilution) was performed over night at 4 °C. Membranes were washed 3 × 5 min in Tris‐buffered saline (TBS) pH 7.6 with 0.1% Tween 20, then incubated with HRP‐linked anti‐rabbit IgG secondary antibodies (Cat. No. 7074, Cell Signaling Technology, 1/2000 dilution) for 1 h at room temperature and washed 3 × 5 min. Membranes were developed using the Clarity Max Western ECL Substrate (Cat. No. 1705062, Bio‐Rad) or Clarity Western ECL Substrate (Cat. No. 1705061, Bio‐Rad), and scanned on the ChemiDocTM MP (Bio‐Rad) Imaging System.