Spa 800

To determine the monomer/dimer ratio of HSPB1, we first separated mitochondria from cytosol using the standard mitochondrial isolation protocol as described above. Samples were kept on ice all the time, and equal amounts of protein lysate were supplemented with NuPAGE LDS Sample buffer (NP0007, Life Technologies) either with or without 100 mM DTT (20290, Thermo Fisher Scientific). The non-reducing condition retained the disulphide bond between HSPB1 dimers and was visualized using the standard SDS–PAGE protocol using anti-HSPB1 (1:1,000, SPA-800, Enzo Life Sciences), Tubulin (1:10,000, ab7291, Abcam) and VDAC1 (1:1,000, ab14734, Abcam).

To assess the phosphorylation of HSPB1, we extracted proteins from untreated cells, cells treated with 10 μM SB203580 (S8307, Sigma-Aldrich), or purified mitochondria with RIPA lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM sodium fluoride, 1 mM sodium pyrophosphate and 0.5% Nonidet-P-40) supplemented by complete protease inhibitor and Phospho-STOP inhibitor mix (4906837001, Roche Applied Science) for 20 min on ice and cleared by centrifugation for 10 min at 20,000g. Samples were kept on ice all the time, and equal amounts of protein lysate were analysed by SDS–PAGE followed by immunoblotting as described above. Primary antibodies were obtained from: HSPB1 (1:1,000, SPA-800, Enzo Life Sciences), pHSPB1 phospho-ser15 (1:500, A00343, Genscript Corporation), pHSPB1 phospho-ser78 (1:500, A00528, Genscript Corporation), pHSPB1 phospho-ser82 (1:500, A00530, Genscript Corporation), pHSPB1 phospho-ser15 (1:1,000, ab5581, Abcam), pHSPB1 phospho-ser78 (1:1,000, ab32501, Abcam), pHSPB1 phospho-ser82 (1:1,000, ab155987, Abcam), Tubulin (1:10,000, ab7291, Abcam), VDAC1 (1:1,000, ab14734, Abcam) and FLAG (1:1,000, F7424, Sigma-Aldrich).