Protein Extraction and Immunoblotting Protocol

Cells were harvested and lysed using modified RIPA buffer (50 mM Tris–HCl at pH 7.5, 150-mM NaCl, 1% Triton × 100, 1-mM EDTA, 0.5% sodium deoxycholate and 0.1% SDS). Cell lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific GmbH, Driesch, Germany) and equal amounts of protein (30 μg) were resolved on 8%–12% polyacrylamide gels before using transferred to polyvinylidene fluoride membranes (EMD Millipore Corporation, Billerica, MA, USA) as previously described^{53 (link)}. After blocking, membranes were blotted with the corresponding primary and horseradish peroxidase-linked secondary antibodies^{54 (link)}. The primary antibodies used were as follows: β-actin (ab8224), eIF2a (ab169528), GADD34 (ab9869) and ATF4 (ab85049) (all from Abcam, USA). CHOP (2895S), Bax D2E11 (5023T), and BCL2 D55G8 (4223T) were purchased from Cell Signaling, USA; ATF6 (ALX-804-381-C100) was purchased from ANZO, USA. Finally, immunoblots were detected by chemiluminescence using the Chemidoc MP system (Bio-Rad, USA). ImageJ V1.49O software (https://imagej.nih.gov/ij/) was used to quantify the immunoblot signals as the mean value of the gray/white scale of all pixels in a band^{55 (link)}.