Anti hsp60

After treatment with IL6 or IL6+NAC, MFE-296 cells were fixed in 4% paraformaldehyde dissolved in PBS. Cells were permeabilized using 0.1% Triton X-100 on ice for 30 minutes; then, cells were blocked with 4% bovine serum for 1 hour at room temperature. Then, cells were incubated with primary anti-dsDNA (1 : 1000, Sigma) and anti-Hsp60 (1 : 1000, Proteintech), for 3 hours at room temperature, followed by incubation with FITC-labeled anti-IgG (1 : 500, Sigma) and Cy3-labeled secondary antibodies for an hour. Immunostaining images were collected using a confocal microscopy. Antibodies used to stain dsDNA have been validated in a previous study [20 (link)].

Reagents used in this study include the following: Methylthiazolyldiphenyl-tetrazolium bromide (MTT)(S6821), PKI-402(S2739), cycloheximide (CHX)(NSC-185) and Thapsigargin (Tg) (S7895)were commercially sourced from Selleckchem (Houston, TX, USA), anti-G3BP1 (13057-2-AP), anti-eIF2α (11233-1-AP), anti-4EBP1 (60246-1-Ig), anti-caspase-9(66169-1-Ig), anti-Bcl-2 (26593-1-AP), anti-Bax (50599-2-Ig), anti-Hsp60 (15282-1-AP), anti-Lonp (66043-1-Ig), anti-Clpp (15698-1-AP), anti-Trap1 (10325-1-AP), anti-Actin(HRP-60008), anti-VDAC (10866-1-AP) (Proteintech, Chicago, IL, USA), anti-YB-1(sc-101198), anti-ATF5(sc-377168), anti-p-Akt(Ser473) (sc-101629)(Santa Cruz, CA, USA), p- eIF2α(Ser51)(3398), p-4EBP1(Thr37/46)(2855), p-Akt(Thr308) (13038), p-P70S6K(9204) (Cell Signaling Technology, USA).

Proteins were extracted with RIPA lysis buffer (CST, Danvers, MA, United States) supplemented with a protease-inhibitor cocktail (Sigma, MO, United States). Proteins were separated by SDS-PAGE, and were blotted with antibodies listed as follows: anti-VDAC (1:1,000, Cat#: ab14734, Abcam, United Kingdom) and anti-GRP75 (1:1,000, Cat#: 14887-1-AP, Proteintech, China), anti-HSP60 (1:1,000, Cat#: 15282-1-AP, Proteintech, China), anti-LONP1 (1:1,000, Cat#: 15440-1-AP, Proteintech, China), and anti-CLPP (1:1,000, Cat#: 15698-1-AP, Proteintech, China). Beta Actin (1:1,000, Cat#: 20536-1-AP, Proteintech, China) antibody was used as an internal antibody.