S0821

For biochemical assays, 5–10 adult fly heads were lysed in immunoprecipitation lysis buffer (NaCl 150 mM, 0,1% Tween-20 (Polyoxyethylene sorbitane monolaureate), TBS pH 7.5). We incubated Protein A/G agarose beads overnight at 4°C with 2 μl of the indicated antibody or control IgG (1:100), followed by incubation at 4°C for 1 h with supernatants. We washed the beads and resuspended in 1× SDS–PAGE loading buffer for western blot analysis in a 4%–12% gradient SDS-PAGE for the detection of sHSP23 and sHSP26. After electro-blotted onto nitrocellulose 0.45 μM (GE Healthcare) 100V for 1 hour, we blocked the membranes in TBS-Tween-20 buffer with 5% BSA. We incubated the membranes overnight at 4°C in constant agitation with anti-Hsp23 antibody (1:1000) (Sigma-Aldrich S0821), anti-Hsp26 (1:1000) (Abmart) We visualized the antibody-protein interaction by chemoluminescence using IRDye Secondary Antibodies anti-mouse (IRDye 800CW, LI-COR), anti-rabbit (IRDye 680 RD, LI-COR) and developed with Odyssey equipment. We used three RNAi tools to downregulate pkm expression to replicate this condition. pkm RNAi 2 was selected to do the rest of experiments due to the evidences we obtained in the blot assay.