Goat anti hsp60 sc 1052

For immunostaining, cells were fixed with 4% paraformaldehyde in PBS, permeabilized and blocked with PBS containing 0.5% Triton X-100 and 3% BSA for 30 min at room temperature. The primary antibodies used were mouse anti-HA (sc-7392; Santa Cruz Biotechnology, Dallas, TX, USA, 1:200 dilution), mouse anti-p62 (ab56416; Abcam, Cambridge, UK, 1:500 dilution), rabbit anti-Tom20 (sc-11415; Santa Cruz Biotechnology, 1:200 dilution) and goat anti-hsp60 (sc-1052; Santa Cruz Biotechnology, 1:200 dilution). The secondary antibodies used were Alexa Fluor dye-conjugated donkey anti-mouse, donkey anti-goat and donkey anti-rabbit (Thermo Scientific, Waltham, MA, USA 1:200 dilution). Nuclei were counterstained with Hoechst 33342 (Thermo Scientific). All fluorescent images were acquired on Yokogawa CSU (confocal scanner unit)-W1 spinning disc system (Andor, Belfast, UK) equipped with a 60 × 1.49 NA objective (Nikon, Tokyo, Japan). The brightness of the images was adjusted using NIS-Elements (Nikon).

Cells were lysed in RIPA (Radioimmunoprecipitation assay) buffer (Sigma-Aldrich, Saint-Quentin-Fallavier, France) containing protease and phosphatase inhibitors (Roche, Meylan, France). Lysates were quantified using the BCA (Bicinchoninic acid) protein assay kit (Thermofisher Scientific). Proteins were separated by SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Biorad, Marnes-la-Coquette, France). The membranes were blocked with 5% fat free milk or Bovine Serum Albumin (BSA, in case of phospho protein) in Tris-buffered Saline (TBS) containing 0.1% Tween-20 (TBS-T) and subsequently incubated with their respective primary antibodies overnight at 4 °C. After washing with TBS-T, membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 45 min, followed by washing with TBS-T. Protein bands were visualized with Amersham ECL substrates (GE Healthcare, Buc, France).
The following antibodies were used: goat anti-HSP60 (sc-1052, Santa Cruz Biotechnology Inc., Heidelberg, Germany), rabbit anti-cleaved Caspase 3 (Asp175) (#9661, Cell Signaling Technology, Saint-Cyr-L’École, France), rabbit anti-Bim (#2933, Cell Signaling Technology), rabbit anti-PTEN (#9559, Cell Signaling Technology), rabbit anti-pAKT (#4058, Cell Signaling Technology), rabbit anti-AKT (#9272, Cell Signaling Technology).