Pierce microplate bca protein assay kit

The hippocampus was homogenized as previously described [43 (link)] with some modifications. Briefly, we used 120 μl of TBS buffer (20 mM Tris-HCl, 137 mM NaCl, pH 7.6) containing protease and phosphatase inhibitors and 1% Triton-X100 in a dounce homogenizer. After 30-min incubation on ice, it was centrifuged at 14000g at 4 °C for 30 min. The supernatant was collected. Protein concentrations were determined (Pierce microplate BCA Protein Assay kit, thermofisher.com). Western blot was used as previously described [44 (link)]. The levels of the synaptic proteins PSD-95 (1:3000, MAB1596, Millipore) and synaptophysin (1:1000, Ab14692, Abcam,) were measured and normalized to beta-actin.

For total protein extraction, mouse liver tissues and cells were homogenized in M-PER™ Mammalian Protein Extraction Reagent (Cat#78501, Thermo Fisher Scientific) containing the Halt™ Protease Inhibitor Cocktail (Cat#78429, Thermo Fisher Scientific). Subsequently, protein concentration was determined using the Pierce™ Microplate BCA Protein Assay Kit (Cat#23252, Thermo Fisher Scientific). For Western blotting, extracted proteins were boiled in Tris-Glycine SDS Sample Buffer (Bio-Rad Laboratories, Inc., Hercules, CA) for denaturation and subsequently separated by SDS-PAGE, and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc.) by electroblotting. Membranes were blocked in 10% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and then incubated with primary antibodies (summarized in Supplementary Table 1) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase-secondary antibody (1:5000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. After appropriate washing, membranes were developed with the Clarity^TM Western ECL Substrate (Cat#170-5061, Bio-Rad Laboratories, Inc.) or Clarity^TM Max Western ECL Substrate (Cat#170-5062, Bio-Rad Laboratories, Inc.).

Protein concentrations in whole-cell lysates were quantified via bicinchoninic acid (BCA) assay using a Pierce Microplate BCA Protein Assay kit (Thermo Scientific, USA). Protein expression was verified via separation in 7.5% (HC1), 10% (LIC2, mTOR, AMBRA1, β-actin and NFKB1) or 12.5% SDS-polyacrylamide gels (LC3, LC8-1/2 and Bim). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The samples were blocked with 5% bovine serum albumin (BSA) in tris-buffered saline with tween 20 (TBS-T) for 1 h and then incubated with the respective primary antibodies using GAPDH as an endogenous control followed by HRP secondary antibody incubation. Proteins were revealed via chemiluminescence with a homemade recipe (Tris 1.5 M pH 8.9, p-coumaric acid 20 mM, luminol 125 mM and hydrogen peroxide 30% in water).
Western blotting images were captured using an ImageQuant LAS 4000 (GE Healthcare, USA) and minimally processed (equally for all samples) using Windows Live software for Windows7 (Microsoft, Redmond, WA, USA). No positive controls, such as proteasome inhibitors, were used in the protein expression analysis due to the lack of information regarding their action on dynein. The analysis compared Amblyomin-X-treated with non-treated cells. The positive controls used for other targets were MG-132 and rapamycin (only for mTOR, AMBRA1 and LC3).