Adi spa 801

Standard Western blotting methods were used. Twenty micrograms of total worm lysate from disruption of 800–1 000 whole worms described above were boiled in Laemmli buffer and loaded onto a gradient SDS-PAGE gel, separated, and transferred onto PVDF membrane. Membranes were stained with LI-COR Total Protein Stain and imaged using the LI-COR Odyssey. Then, membranes were probed with anti-HspB1 antibody (ADI-SPA-801, ENZO Life Sciences, Farmingdale, NY;1:1 000 overnight at 4°C), followed by a fluorescent secondary antibody and imaging on the LI-COR Odyssey and analysis on LI-COR Imaging software.

The expression levels of various HSPs were probed with specific antibodies in GPI-mGFP–expressing CHO cells, as follows: HSP25 (ADI-SPA-801, Enzo Life Sciences, Inc., Farmingdale, NY), HSP70 (ADI-SPA-810, Enzo), HSP90 (SMC-107, StressMarq Biosciences, Victoria, BC, Canada), HSP60 (SPC-105, StressMarq), and GRP78 (SPC-180, StressMarq). For the analysis of protein phosphorylation, specific antibodies against phospho-HSP25 (Ser86) (44536, Invitrogen, Carlsbad, CA), phospho-HSP27 (Ser15) (SPA-525, Stressgen, San Diego, CA), phospho-HSF1 (Ser326) (ADI-SPA-902, Enzo), and HSF1 (RT-405, Thermo Scientific, Inc., Waltham, MA) were used. The cells were harvested and lysed in Laemmli sample buffer. Equal protein amounts were measured using Pierce 660 nm protein assay (22660, Thermo Scientific) and loaded on 8% (for HSF1) or 10% SDS gels (for all other determinations) for western blotting experiments⁷¹ Enhanced chemiluminescence was detected and analyzed using Alpha Ease FC Software v6 (San Jose, CA, USA) after washing and incubating with secondary antibodies IgG peroxidase conjugate (anti-mouse A3682, anti-rabbit A9169, Sigma- Aldrich, Saint Louise, MO). All dilutions were carried out based on the manufacturers' recommendations. Anti-GAPDH (G9545, Sigma-Aldrich) antibody was used to control the protein load and for data normalization.