Compat able bca protein assay kit

Total protein was prepared using RIPA lysis buffer (Beyotime Biotech). Cell culture supernatant was collected and concentrated using a Microcon-10 kDa Centrifugal Filter Unit with Ultracel (Millipore). The protein content was measured using a Compat-Able BCA Protein Assay Kit (Thermo Scientific). Proteins were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad). The membranes were blocked with PBS containing 5% milk and 0.1% Tween-20 at room temperature for 1 h. The membranes were then immunoblotted with the desired primary antibodies and incubated with the corresponding horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Table S2). Immunoreactive bands were visualized using an Amersham ECL Western Blotting Detection Kit according to the manufacturer's instructions. β-Actin served as a loading control.

Total RNA was extracted from cells using Laemmli sample buffer with 5% 2-mercaptoethanol (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA). The Compat-Able™ BCA protein assay kit (cat no. 23229; Thermo Fisher Scientific, Inc.) was used to perform protein quantitation. A total of 40 µg protein per lane was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Blocking of the membranes was performed using 5% non-fat milk or 3% bovine serum albumin (cat no. A500023-0100; Sangon Biotech Co., Ltd., Shanghai, China). Primary antibodies used were ATF4 (cat no. ab50546; 1:5,000) and MYC (cat no. ab32072; 1:5,000) (both from Abcam, Cambridge, UK). GAPDH (cat no. G9545; 1:5,000; Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) was used as normalization control. Primary antibodies were incubated with the membrane for 8 h at 4°C. The membranes were washed three times with Tris-buffered saline with 0.2% Tween-20 and incubated with goat anti-mouse (cat no. ab6789; 1:2,000) and goat anti-rabbit secondary antibodies (cat no. ab6721; 1:2,000) (both from Abcam) labeled with horseradish peroxidase >1 h at room temperature, and signals were visualized using film exposure. All experiments were performed in triplicate.