Trans blot turbo mini 0.2 μm nitrocellulose membranes

Proteins were extracted from cell lines using mammalian protein extraction reagent (MPER; 50 nM Tris-HCl, 200 mM NaCl, 0.25% Triton 100X, and 10% glycerol) containing a protease and phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA, USA; PIA32961). Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA; 500–0006). Twenty micrograms of protein were separated in precast 4–15% gradient Tris-glycine SDS-polyacrylamide gels (Mini-PROTEAN^® TGXTM Precast Gels, Bio-Rad, Hercules, CA, USA; 456–1086) and transferred in Trans-Blot Turbo Mini 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA; 170–4159). Membranes were blocked with 5% milk in PBS-Tween for 1 h and incubated overnight at 4 °C with primary antibody in BSA 1%. Membranes were next incubated with secondary antibody for 1 h, and signal was detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). ß-Actin was used as a loading protein control and each experiment was repeated three times. Image J [20 (link)] was used to quantify protein levels in Western blots.

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.