Anti clpp

Western blotting analysis was performed as detailed elsewhere (42 (link),44 (link),49 (link)). Fish were sacrificed after anesthesia and homogenized in RIPA reagent (Invitrogen) using a homogenizer. Twenty micrograms of total cellular proteins were electrophoresed through 10% bis-Tris SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane. The antibodies used include anti-Gtpbp3 (Sigma, HPA042158), anti-Nd5 (Abcam, ab92624), anti-Nd6 (Abcam, ab81212), anti-Cytb (ABclonal, A9762), anti-Co2 (Proteintech, 55070-1-AP), anti-Sdha (Proteintech, 14865-1-AP), anti-Atp5c (Proteintech 60284-1-Ig), anti-Tfam (Proteintech, 19998-1-AP), and anti-Tufm (Proteintech, 26730-1-AP), anti-Yars2 (Abcam, ab228957), anti-Kars (Proteintech, 14951-1-AP), anti-Afg3l2 (Proteintech, 14631-1-AP), anti-Clpp (Proteintech, 15698-1-AP) and anti-Gapdh (Sigma, SAB2701826). Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO). Quantification of density in each band was performed as detailed previously (42 (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.