Pro q diamond staining

Protein extraction was performed within a week after PZQ treatment, using the frozen extraction method and phosphoprotein enrichment was performed using the TALON PMAC Magnetic Phospho Enrichment Kit (Takara, Shiga, Japan). First, 10 pairs of untreated and 40 μg/mL-PZQ-treated worms were put into a mortar and homogenized in liquid nitrogen. After the nitrogen had completely evaporated, lysis buffer (provided with the kit) was added and incubated on ice for 10 min. Protein lysate was transferred into tubes and centrifuged at 5000 × g for 5 min at 4 °C. The supernatant was transferred to new tube and the protein concentration was measured using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA).
Phosphoprotein enrichment was performed according to manufacturer’s protocol. Briefly, 250 μg protein was mixed with metal ion-coated magnetic beads and mixed on a rotary shaker for 90 min at 4 °C. The supernatant was discarded, and proteins bound to beads were eluted using 75 μL of the provided elution buffer. Successful enrichment was confirmed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Pro-Q diamond staining (Thermo Fisher Scientific). All experiments were performed in three biologic replicates.

Titin isoform expression was determined by large format 1% sodium-dodecyl-sulfate (SDS)—agarose gel electrophoresis [40 (link)]. Gel electrophoresis was performed at 16 mA/gel for 3 h 30 min. Gels were stained with SYPRO Ruby Protein Gel Stain (Thermo Fischer Scientific, Waltham, MA, USA), which gives the maximum signal strength and widest linear dynamic range for protein quantification [41 (link)]. Gels were scanned by using a Typhoon laser-scanner (Amersham BioSciences, Little Chalfont, Buckinghamshire, United Kingdom), and optical density was analyzed with ImageJ software (ImageJ 1.52k, National Institutes of Health, Bethesda, MD, USA). Titin isoform ratio (N2BA/N2B) was calculated from the integrated band densities. Relative content of total titin (TT) was normalized to myosin heavy chain (MHC), and T2 (titin’s proteolytic degradation product) was normalized to TT. The two N2BA bands were summed to determine the relative N2BA isoform content [24 (link),42 (link)]. To determine total titin phosphorylation, 1% SDS-agarose gel electrophoresis was performed. Gels were stained for phosphoprotein using Pro-Q Diamond staining (Thermo Fisher Scientific, Waltham, MA, USA) and scanned with a Typhoon laser scanner. Gels were then stained for total protein content with SYPRO Ruby Protein Gel Stain and scanned with a Typhoon laser scanner. Bands were quantified with the ImageJ software.