Ab109376

Tissue homogenates and cells were treated with RIPA lysates (Beyotime, China), and supernatants containing proteins were collected by centrifugation. Protein content was assessed by BCA detection kit (EMD Millipore). SDS-PAGE was performed with the same amount of protein samples in each lane, and then the isolated proteins were electrotransferred to PVDF membrane (Millipore). 5% skimmed milk powder was used for blocking the membranes, and then the corresponding primary antibody (eIF5A (1 : 1000, ab32443, Abcam); FANCD2 (1 : 1000, ab108928, Abcam); SLC7A11 (1 : 1000, ab216876, Abcam); HSPB1 (1 : 1000, ab109376, Abcam); Bax (1 : 1000, ab53154, Abcam); Bcl-2 (1 : 1000, ab32124, Abcam); cleaved caspase-3 (1 : 1000, ab32042, Abcam); cytochrome C (1 : 1000, ab133504, Abcam); β-actin (1 : 1000, ab8226, Abcam)) was applied overnight at 4°C. Following that, the membranes were treated for 2 h with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L (1 : 2000, ab6721, Abcam) and Rabbit Anti-Mouse IgG H&L (1 : 2000, ab6728, Abcam)). The transfer protein on membranes was developed with electrochemiluminescence (ECL, Thermo Fisher Scientific, USA)). Grayscale of the strips was assessed by ImageJ 1.48v software (NIH).

Slices were dewaxed and hydrated. Endogenous catalase was removed by H₂O₂. The slices were blocked for 30 min at 37°C with 5% BSA solution (P0220, Beyotime). Following that, the sections were reacted with primary antibody [eIF5A (1 : 250, ab32443, Abcam); FANCD2 (1 : 100, ab108928, Abcam); SLC7A11 (1 : 500, ab216876, Abcam); HSPB1 (1 : 500, ab109376, Abcam)] overnight at 4°C. The sections were incubated with the secondary antibody for 30 min. Then the slides were treated for 5 min with diaminobenzidine (DAB, Beyotime, China). After restaining for 5 min with hematoxylin, the slices were dehydrated, made transparent, and finally sealed with neutral resin. The results were observed with an optical microscope (Olympus Corporation).

Post lentivirus infection, cells in stable growth were harvested for protein extraction using the BCa protein assay kit (HyClone-Pierce, #23225). Proteins were resolved on a 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 1 hour, incubated with primary antibodies overnight at 4°C, and then with secondary antibodies for 1 hour. After washing thrice with TBST, protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, #RPN2232) and captured using a chemiluminescence imager. Primary antibodies included HSPB8 (1:1000, Proteintech, 15287-1-AP), PRAS40 (1:1000, Abcam, #ab151719), p-PRAS40 (1:1000, Abcam, # ab134084), HSP27 (1:1000, Abcam, #ab109376), p-HSP27 (1:1000, Abcam, # ab155987), and GAPDH (1:30000, Proteintech, #60004-1-lg). Secondary antibodies were goat anti-mouse (1:3000, Beyotime, #A0216) and goat anti-rabbit (1:3000, Beyotime, #A0208).